Archive for the ‘Ionic Transporters Na+’ Category

Summary of Lipid Metabolism


Author: Larry H. Bernstein, MD, FCAP


Lipid Classification System

The LIPID MAPS Lipid Classification System is comprised of eight lipid categories, each with its own sublassification hierarchy.

Each LMSD record contains an image of the

  • molecular structure,
  • common and systematic names,
  • links to external databases,
  • Wikipedia pages (where available),
  • other annotations and links to structure viewing tools.

All lipids in the LIPID MAPS Structure Database (LMSD) have been classified using this system and have been assigned LIPID MAPS ID’s (LM_ID) which reflects their position in the classification hierarchy.

The LIPID MAPS Structure Database (LMSD) is a relational database encompassing structures and annotations of biologically relevant lipids. As of May 3, 2013, LMSD contains over 37,500 unique lipid structures, making it the largest public lipid-only database in the world. Structures of lipids in the database come from several sources:

  • LIPID MAPS Consortium’s core laboratories and partners;
  • lipids identified by LIPID MAPS experiments;
  • biologically relevant lipids manually curated from LIPID BANK, LIPIDAT, Lipid Library, Cyberlipids, ChEBI and other public sources;
  • novel lipids submitted to peer-reviewed journals;
  • computationally generated structures for appropriate classes.

All the lipid structures in LMSD adhere to the structure drawing rules proposed by the LIPID MAPS consortium. A number of structure viewing options are offered: gif image (default), Chemdraw (requires Chemdraw ActiveX/Plugin), MarvinView (Java applet) and JMol (Java applet).

(as of 10/8/14)

Number of lipids per category

Fatty acyls          5869

Glycerolipids       7541

Glycerophospholipids       8002

Sphingolipids      4338

Sterol lipids         2715

Prenol lipids        1259

Sacccharolipids  1293

Polyketides         6742

TOTAL  37,759 structures


Sud M, Fahy E, Cotter D, Brown A, Dennis EA, Glass CK, Merrill AH Jr, Murphy RC, Raetz CR, Russell DW, Subramaniam S. LMSD: LIPID MAPS structure database Nucleic Acids Research 35: p. D527-32. PMID:17098933 [doi:10.1093/nar/gkl838] PMID: 17098933

Fahy E, Sud M, Cotter D & Subramaniam S. LIPID MAPS online tools for lipid research Nucleic Acids Research (2007) 35: p. W606-12.PMID:17584797 [doi:10.1093/nar/gkm324] PMID: 17584797 

The Recognition of Essential Fatty Acids

Dietary fat has long been recognized as an important source of energy for mammals, but in the late 1920s, researchers demonstrated the dietary requirement for particular fatty acids, which came to be called essential fatty acids. It was not until the advent of intravenous feeding, however, that the importance of essential fatty acids was widely accepted: Clinical signs of essential fatty acid deficiency are generally observed only in patients on total parenteral nutrition who received mixtures devoid of essential fatty acids or in those with malabsorption syndromes.

These signs include dermatitis and changes in visual and neural function. Over the past 40 years, an increasing number of physiological functions, such as immunomodulation, have been attributed to the essential fatty acids and their metabolites, and this area of research remains quite active.1, 2

Fatty Acid Nomenclature

The fat found in foods consists largely of a heterogeneous mixture of triacylglycerols (triglycerides)–glycerol molecules that are each combined with three fatty acids. The fatty acids can be divided into two categories, based on chemical properties: saturated fatty acids, which are usually solid at room temperature, and unsaturated fatty acids, which are liquid at room temperature. The term “saturation” refers to a chemical structure in which each carbon atom in the fatty acyl chain is bound to (saturated with) four other atoms, these carbons are linked by single bonds, and no other atoms or molecules can attach; unsaturated fatty acids contain at least one pair of carbon atoms linked by a double bond, which allows the attachment of additional atoms to those carbons (resulting in saturation). Despite their differences in structure, all fats contain approximately the same amount of energy (37 kilojoules/gram, or 9 kilocalories/gram).

The class of unsaturated fatty acids can be further divided into monounsaturated and polyunsaturated fatty acids. Monounsaturated fatty acids (the primary constituents of olive and canola oils) contain only one double bond. Polyunsaturated fatty acids (PUFAs) (the primary constituents of corn, sunflower, flax seed and many other vegetable oils) contain more than one double bond. Fatty acids are often referred to using the number of carbon atoms in the acyl chain, followed by a colon, followed by the number of double bonds in the chain (e.g., 18:1 refers to the 18-carbon monounsaturated fatty acid, oleic acid; 18:3 refers to any 18-carbon PUFA with three double bonds).

PUFAs are further categorized on the basis of the location of their double bonds. An omega or n notation indicates the number of carbon atoms from the methyl end of the acyl chain to the first double bond. Thus, for example, in the omega-3 (n-3) family of PUFAs, the first double bond is 3 carbons from the methyl end of the molecule.  Finally, PUFAs can be categorized according to their chain length. The 18-carbon n-3 and n-6 short-chain PUFAs are precursors to the longer 20- and 22-carbon PUFAs, called long-chain PUFAs (LCPUFAs).

Fatty Acid Metabolism

Mammalian cells can introduce double bonds into all positions on the fatty acid chain except the n-3 and n-6 position. Thus, the short-chain alpha- linolenic acid (ALA, chemical abbreviation: 18:3n-3) and linoleic acid (LA, chemical abbreviation: 18:2n-6) are essential fatty acids.

No other fatty acids found in food are considered ‘essential’ for humans, because they can all be synthesized from the short chain fatty acids.

Following ingestion, ALA and LA can be converted in the liver to the long chain, more unsaturated n-3 and n-6 LCPUFAs by a complex set of synthetic pathways that share several enzymes (Figure 1). LC PUFAs retain the original sites of desaturation (including n-3 or n-6). The omega-6 fatty acid LA is converted to gamma-linolenic acid (GLA, 18:3n-6), an omega- 6 fatty acid that is a positional isomer of ALA. GLA, in turn, can be converted to the longerchain omega-6 fatty acid, arachidonic acid (AA, 20:4n-6). AA is the precursor for certain classes of an important family of hormone- like substances called the eicosanoids (see below).

The omega-3 fatty acid ALA (18:3n-3) can be converted to the long-chain omega-3 fatty acid, eicosapentaenoic acid (EPA; 20:5n-3). EPA can be elongated to docosapentaenoic acid (DPA 22:5n-3), which is further desaturated to docosahexaenoic acid (DHA; 22:6n-3). EPA and DHA are also precursors of several classes of eicosanoids and are known to play several other critical roles, some of which are discussed further below.

The conversion from parent fatty acids into the LC PUFAs – EPA, DHA, and AA – appears to occur slowly in humans. In addition, the regulation of conversion is not well understood, although it is known that ALA and LA compete for entry into the metabolic pathways.

Physiological Functions of EPA and AA

As stated earlier, fatty acids play a variety of physiological roles. The specific biological functions of a fatty acid are determined by the number and position of double bonds and the length of the acyl chain.

Both EPA (20:5n-3) and AA (20:4n-6) are precursors for the formation of a family of hormone- like agents called eicosanoids. Eicosanoids are rudimentary hormones or regulating – molecules that appear to occur in most forms of life. However, unlike endocrine hormones, which travel in the blood stream to exert their effects at distant sites, the eicosanoids are autocrine or paracrine factors, which exert their effects locally – in the cells that synthesize them or adjacent cells. Processes affected include the movement of calcium and other substances into and out of cells, relaxation and contraction of muscles, inhibition and promotion of clotting, regulation of secretions including digestive juices and hormones, and control of fertility, cell division, and growth.3

The eicosanoid family includes subgroups of substances known as prostaglandins, leukotrienes, and thromboxanes, among others. As shown in Figure 1.1, the long-chain omega-6 fatty acid, AA (20:4n-6), is the precursor of a group of eicosanoids that include series-2 prostaglandins and series-4 leukotrienes. The omega-3 fatty acid, EPA (20:5n-3), is the precursor to a group of eicosanoids that includes series-3 prostaglandins and series-5 leukotrienes. The AA-derived series-2 prostaglandins and series-4 leukotrienes are often synthesized in response to some emergency such as injury or stress, whereas the EPA-derived series-3 prostaglandins and series-5 leukotrienes appear to modulate the effects of the series-2 prostaglandins and series-4 leukotrienes (usually on the same target cells). More specifically, the series-3 prostaglandins are formed at a slower rate and work to attenuate the effects of excessive levels of series-2 prostaglandins. Thus, adequate production of the series-3 prostaglandins seems to protect against heart attack and stroke as well as certain inflammatory diseases like arthritis, lupus, and asthma.3.

EPA (22:6 n-3) also affects lipoprotein metabolism and decreases the production of substances – including cytokines, interleukin 1ß (IL-1ß), and tumor necrosis factor a (TNF-a) – that have pro-inflammatory effects (such as stimulation of collagenase synthesis and the expression of adhesion molecules necessary for leukocyte extravasation [movement from the circulatory system into tissues]).2 The mechanism responsible for the suppression of cytokine production by omega-3 LC PUFAs remains unknown, although suppression of omega-6-derived eicosanoid production by omega-3 fatty acids may be involved, because the omega-3 and omega-6 fatty acids compete for a common enzyme in the eicosanoid synthetic pathway, delta-6 desaturase.

DPA (22:5n-3) (the elongation product of EPA) and its metabolite DHA (22:6n-3) are frequently referred to as very long chain n-3 fatty acids (VLCFA). Along with AA, DHA is the major PUFA found in the brain and is thought to be important for brain development and function. Recent research has focused on this role and the effect of supplementing infant formula with DHA (since DHA is naturally present in breast milk but not in formula).

Overview of Lipid Catabolism:

The major aspects of lipid metabolism are involved with

  • Fatty Acid Oxidation to produce energy or
  • the synthesis of lipids which is called Lipogenesis.

The metabolism of lipids and carbohydrates are related by the conversion of lipids from carbohydrates. This can be seen in the diagram. Notice the link through actyl-CoA, the seminal discovery of Fritz Lipmann. The metabolism of both is upset by diabetes mellitus, which results in the release of ketones (2/3 betahydroxybutyric acid) into the circulation.


fatty acid metabolism

fatty acid metabolism

The first step in lipid metabolism is the hydrolysis of the lipid in the cytoplasm to produce glycerol and fatty acids.

Since glycerol is a three carbon alcohol, it is metabolized quite readily into an intermediate in glycolysis, dihydroxyacetone phosphate. The last reaction is readily reversible if glycerol is needed for the synthesis of a lipid.

The hydroxyacetone, obtained from glycerol is metabolized into one of two possible compounds. Dihydroxyacetone may be converted into pyruvic acid, a 3-C intermediate at the last step of glycolysis to make energy.

In addition, the dihydroxyacetone may also be used in gluconeogenesis (usually dependent on conversion of gluconeogenic amino acids) to make glucose-6-phosphate for glucose to the blood or glycogen depending upon what is required at that time.

Fatty acids are oxidized to acetyl CoA in the mitochondria using the fatty acid spiral. The acetyl CoA is then ultimately converted into ATP, CO2, and H2O using the citric acid cycle and the electron transport chain.

There are two major types of fatty acids – ω-3 and ω-6.  There are also saturated and unsaturated with respect to the existence of double bonds, and monounsaturated and polyunsatured.  Polyunsaturated fatty acids (PUFAs) are important in long term health, and it will be seen that high cardiovascular risk is most associated with a low ratio of ω-3/ω-6, the denominator being from animal fat. Ω-3 fatty acids are readily available from fish, seaweed, and flax seed. More can be said of this later.

Fatty acids are synthesized from carbohydrates and occasionally from proteins. Actually, the carbohydrates and proteins have first been catabolized into acetyl CoA. Depending upon the energy requirements, the acetyl CoA enters the citric acid cycle or is used to synthesize fatty acids in a process known as LIPOGENESIS.

The relationships between lipid and carbohydrate metabolism are
summarized in Figure 2.

fatty acid spiral


Energy Production Fatty Acid Oxidation:

Visible” ATP:

In the fatty acid spiral, there is only one reaction which directly uses ATP and that is in the initiating step. So this is a loss of ATP and must be subtracted later.

A large amount of energy is released and restored as ATP during the oxidation of fatty acids. The ATP is formed from both the fatty acid spiral and the citric acid cycle. 

Connections to Electron Transport and ATP:

One turn of the fatty acid spiral produces ATP from the interaction of the coenzymes FAD (step 1) and NAD+ (step 3) with the electron transport chain. Total ATP per turn of the fatty acid spiral is:

Electron Transport Diagram – (e.t.c.)

Step 1 – FAD into e.t.c. = 2 ATP
Step 3 – NAD+ into e.t.c. = 3 ATP
Total ATP per turn of spiral = 5 ATP

In order to calculate total ATP from the fatty acid spiral, you must calculate the number of turns that the spiral makes. Remember that the number of turns is found by subtracting one from the number of acetyl CoA produced. See the graphic on the left bottom.

Example with Palmitic Acid = 16 carbons = 8 acetyl groups

Number of turns of fatty acid spiral = 8-1 = 7 turns

ATP from fatty acid spiral = 7 turns and 5 per turn = 35 ATP.

This would be a good time to remember that single ATP that was needed to get the fatty acid spiral started. Therefore subtract it now.

NET ATP from Fatty Acid Spiral = 35 – 1 = 34 ATP

SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver

Jay D. Horton1,2, Joseph L. Goldstein1 and Michael S. Brown1

1Department of Molecular Genetics, and
2Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA

J Clin Invest. 2002;109(9):1125–1131.
Lipid homeostasis in vertebrate cells is regulated by a family of membrane-bound transcription factors designated sterol regulatory element–binding proteins (SREBPs). SREBPs directly activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids, as well as the NADPH cofactor required to synthesize these molecules (14). In the liver, three SREBPs regulate the production of lipids for export into the plasma as lipoproteins and into the bile as micelles. The complex, interdigitated roles of these three SREBPs have been dissected through the study of ten different lines of gene-manipulated mice. These studies form the subject of this review.

SREBPs: activation through proteolytic processing

SREBPs belong to the basic helix-loop-helix–leucine zipper (bHLH-Zip) family of transcription factors, but they differ from other bHLH-Zip proteins in that they are synthesized as inactive precursors bound to the endoplasmic reticulum (ER) (1, 5). Each SREBP precursor of about 1150 amino acids is organized into three domains: (a) an NH2-terminal domain of about 480 amino acids that contains the bHLH-Zip region for binding DNA; (b) two hydrophobic transmembrane–spanning segments interrupted by a short loop of about 30 amino acids that projects into the lumen of the ER; and (c) a COOH-terminal domain of about 590 amino acids that performs the essential regulatory function described below.

In order to reach the nucleus and act as a transcription factor, the NH2-terminal domain of each SREBP must be released from the membrane proteolytically (Figure1). Three proteins required for SREBP processing have been delineated in cultured cells, using the tools of somatic cell genetics (see ref. 5for review). One is an escort protein designated SREBP cleavage–activating protein (SCAP). The other two are proteases, designated Site-1 protease (S1P) and Site-2 protease (S2P). Newly synthesized SREBP is inserted into the membranes of the ER, where its COOH-terminal regulatory domain binds to the COOH-terminal domain of SCAP (Figure 1).

Figure 1

Model for the sterol-mediated proteolytic release of SREBPs from membranes JCI0215593.f1

Model for the sterol-mediated proteolytic release of SREBPs from membranes. SCAP is a sensor of sterols and an escort of SREBPs. When cells are depleted of sterols, SCAP transports SREBPs from the ER to the Golgi apparatus, where two proteases, Site-1 protease (S1P) and Site-2 protease (S2P), act sequentially to release the NH2-terminal bHLH-Zip domain from the membrane. The bHLH-Zip domain enters the nucleus and binds to a sterol response element (SRE) in the enhancer/promoter region of target genes, activating their transcription.

SCAP is both an escort for SREBPs and a sensor of sterols. When cells become depleted in cholesterol, SCAP escorts the SREBP from the ER to the Golgi apparatus, where the two proteases reside. In the Golgi apparatus, S1P, a membrane-bound serine protease, cleaves the SREBP in the luminal loop between its two membrane-spanning segments, dividing the SREBP molecule in half. (Fig 1)  The NH2-terminal bHLH-Zip domain is then released from the membrane via a second cleavage mediated by S2P, a membrane-bound zinc metalloproteinase. The NH2-terminal domain, designated nuclear SREBP (nSREBP), translocates to the nucleus, where it activates transcription by binding to nonpalindromic sterol response elements (SREs) in the promoter/enhancer regions of multiple target genes.

SREBPs: two genes, three proteins

The mammalian genome encodes three SREBP isoforms, designated SREBP-1a, SREBP-1c, and SREBP-2.

SREBP-1a is a potent activator of all SREBP-responsive genes, including those that mediate the synthesis of cholesterol, fatty acids, and triglycerides. High-level transcriptional activation is dependent on exon 1a, which encodes a longer acidic transactivation segment than does the first exon of SREBP-1c. The roles of SREBP-1c and SREBP-2 are more restricted than that of SREBP-1a. SREBP-1c preferentially enhances transcription of genes required for fatty acid synthesis but not cholesterol synthesis.

SREBP-1c and SREBP-2 activate three genes required to generate NADPH, which is consumed at multiple stages in these lipid biosynthetic pathways (8) (Figure 2).

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides


A major class of lipids, steroids, have a ring structure of three cyclohexanes and one
cyclopentane in a fused ring system as shown below. There are a variety of functional
groups that may be attached. The main feature, as in all lipids, is the large number of
carbon-hydrogens which make steroids non-polar.

Steroids include such well known compounds as cholesterol, sex hormones, birth
control pills, cortisone, and anabolic steroids.


sex hormones

sex hormones



Adrenocorticoid Hormones

The adrenocorticoid hormones are products of the adrenal glands.

The most important mineralcorticoid is aldosterone, which regulates the
reabsorption of sodium and chloride ions in the kidney tubules and increases
the loss of potassium ions.Aldosterone is secreted when blood sodium ion
levels are too low to cause the kidney to retain sodium ions. If sodium
levels are elevated, aldosterone is not secreted, so that some sodium
will be lost in the urine. Aldosterone also controls swelling in the tissues.

Cortisol, the most important glucocortinoid, has the function of increasing
glucose and glycogen concentrations in the body. These reactions are
completed in the liver by taking fatty acids from lipid storage cells and
amino acids from body proteins to make glucose and glycogen.

In addition, cortisol is elevated in the circulation with cytokine mediated
(IL1, IL1, TNFα) inflammatory reaction, called the systemic inflammatory
response syndrome. Its ketone derivative, cortisone, has the ability
to relieve inflammatory effects. Cortisone or similar synthetic derivatives
such as prednisolone are used to treat inflammatory diseases, rheumatoid
arthritis, and bronchial asthma. There are many side effects with the use
of cortisone drugs, such as bone resorption, so there use must be
monitored carefully.

Hormone Receptors

Steroid hormone receptors are found on the plasma membrane, in the cytosol and also in the nucleus of target cells. They are generally intracellular receptors (typically cytoplasmic) and initiate signal transduction for steroid hormones which lead to changes in gene expression over a time period of hours to days. The best studied steroid hormone receptors are members of the nuclear receptor subfamily 3 (NR3) that include receptors for estrogen (group NR3A)[1] and 3-ketosteroids (group NR3C).[2] In addition to nuclear receptors, several G protein-coupled receptors and ion channels act as cell surface receptors for certain steroid hormones.


Steroid Hormone Receptors and their Response Elements

Steroid hormone receptors are proteins that have a binding site for a particular steroid molecule. Their response elements are DNA sequences that are bound by the complex of the steroid bound to its Steroid receptor.

The response element is part of the promoter of a gene. Binding by the receptor activates or represses, as the case may be, the gene controlled by that promoter.

It is through this mechanism that steroid hormones turn genes on (or off).

steroid hormone receptor

steroid hormone receptor

The glucocorticoid receptor, like all steroid hormone receptors, is a zinc-finger transcription factor; the zinc atoms are the four yellow spheres. Each is attached to four cysteines.

For a steroid hormone to regulate (turn on or off) gene transcription, its receptor must:

  1. bind to the hormone (cortisol in the case of the glucocorticoid receptor)
  2. bind to a second copy of itself to form a homodimer
  3. be in the nucleus, moving from the cytosol if necessary
  4. bind to its response element
  5. bind to other protein cofactors

Each of these functions depend upon a particular region of the protein (e.g., the zinc fingers for binding DNA).

Each of these functions depend upon a particular region of the protein (e.g., the zinc fingers for binding DNA). Mutations in any one region may upset the function of that region without necessarily interfering with other functions of the receptor.

Positive and Negative Response Elements

Some of the hundreds of glucocorticoid response elements in the human genome activate gene transcription when bound by the hormone/receptor complex. Others inhibit gene transcription when bound by the hormone/receptor complex.

Example: When the stress hormone cortisol — bound to its receptor — enters the nucleus of a liver cell, the complex binds to the positive response elements of the many genes needed for gluconeogenesis — the conversion of protein and fat into glucose resulting in a rise in the level of blood sugar.

the negative response element of the insulin receptor gene thus diminishing the ability of the cells to remove glucose from the blood. (This hyperglycemic effect is enhanced by the binding of the cortisol/receptor complex to a negative response element in the beta cells of the pancreas thus reducing the production of insulin.)

Note that every type of cell in the body contains the same response elements in its genome. What determines if a given cell responds to the arrival of a hormone depends on the presence of the hormone’s receptor in the cell.

The Nuclear Receptor Superfamily



 The zinc-finger proteins that serve as receptors for glucocorticoids and progesterone are members of a large family of similar proteins that serve as receptors for a variety of small, hydrophobic molecules. These include:

  1. other steroid hormones like
  2. the mineralocorticoid aldosterone
  3. estrogens
  4. the thyroid hormone, T3
  5. calcitriol, the active form of vitamin D
  6. retinoids: vitamin A (retinol) and its relatives
    1. retinal
    2. retinoic acid (tretinoin — also available as the drug Retin-A®); and its isomer
  7. isotretinoin (sold as Accutane® for the treatment of acne).
  8. bile acids
  9. fatty acids.

These bind members of the superfamily called peroxisome-proliferator-activated receptors (PPARs). They got their name from their initial discovery as the receptors for

  • drugs that increase the number and size of peroxisomes in cells.

In every case, the receptors consist of at least

  • three functional modules or domains.

From N-terminal to C-terminal, these are:

  1. a domain needed
  2. the zinc-finger domain needed for DNA binding (to the response element)
  3. the domain responsible for binding the particular hormone as well as the second unit of the dimer.
  4. for the receptor to activate the promoters of the genes being controlled

Schematic diagram of type II zinc finger proteins characteristic of the DNA-binding domain structure of members of the steroid hormone receptor superfamily. Zinc fingers are common features of many transcription factors, allowing proteins to bind to DNA. Each circle represents one amino acid. The CI zinc finger interacts specifically with five base pairs of DNA and determines the DNA sequence recognized by the particular steroid receptor. The three shaded amino acids indicated by the arrows in the knuckle of the CI zinc finger are in the “P box” that allows HRE sequence discrimination between the GR and ERα. The vertically striped aa within the knuckle of the CII zinc finger constitutes the “D box” that is important for dimerization and contacts with the DNA phosphate backbone. Adapted from Tsai M-J, O’Malley BW. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 1994;63:451-483; Gronemeyer H. Transcription activation by estrogen and progesterone receptors. Annu Rev Genet 1991;25:89-123.

type II zinc finger proteins

type II zinc finger proteins

Cytoskeleton and Cell Membrane Physiology

Definition and Function

The cytoskeleton is a series of intercellular proteins that help a cell with

  1. shape,
  2. support, and
  3. movement.

Cytoskeleton has three main structural components:

  1. microfilaments,
  2. intermediate filaments, and
  3. movement

The cytoskeleton mediates movement by

  • helping the cell move in its environment and
  • mediating the movement of the cell’s components.

Thereby it provides an important structural framework for the cell –

  • the framework for the movement of organelles, contiguous with the cell membrane, around the cytoplasm. By the activity of
  • the network of protein microfilaments, intermediate filaments, and microtubules.

The structural framework supports cell function as follows:

Cell shape. For cells without cell walls, the cytoskeleton determines the shape of the cell. This is one of the functions of the intermediate filaments.

Cell movement. The dynamic collection of microfilaments and microtubles can be continually in the process of assembly and disassembly, resulting in forces that move the cell. There can also be sliding motions of these structures. Audesirk and Audesirk give examples of white blood cells “crawling” and the migration and shape changes of cells during the development of multicellular organisms.

Organelle movement. Microtubules and microfilaments can help move organelles from place to place in the cell. In endocytosis a vesicle formed engulfs a particle abutting the cell. Microfilaments then attach to the vesicle and pull it into the cell. Much of the complex synthesis and distribution function of the endoplasmic reticulum and the Golgi complex makes use of transport vescicles,  associated with the cytoskeleton.

Cell division. During cell division, microtubules accomplish the movement of the chromosones to the daughter nucleus. Also, a ring of microfilaments helps divide two developing cells by constricting the central region between the cells (fission).

Hickman, et al. Ch 4 Hickman, Cleveland P., Roberts, Larry S., and Larson, Allan, Integrated Principles of Zoology, 9th. Ed., Wm C. Brown, 1995.
Audesirk & Audesirk Ch 6 Audesirk, Teresa and Audesirk, Gerald, Biology, Life on Earth, 5th Ed., Prentice-Hall, 1999.



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Cytoskeleton and Cell Membrane Physiology


Curator: Larry H Bernstein, MD, FCAP




early evolution of lipid membranes and the three domains of life

early evolution of lipid membranes and the three domains of life

Definition and Function

The cytoskeleton is a series of intercellular proteins that help a cell with

  1. shape,
  2. support, and
  3. movement.

Cytoskeleton has three main structural components:

  1. microfilaments,
  2. intermediate filaments, and
  3. movement

The cytoskeleton mediates movement by

  • helping the cell move in its environment and
  • mediating the movement of the cell’s components.

Thereby it provides an important structural framework for the cell –

  • the framework for the movement of organelles, contiguous with the cell membrane, around the cytoplasm. By the activity of
  • the network of protein microfilaments, intermediate filaments, and microtubules.

The structural framework supports cell function as follows:

Cell shape. For cells without cell walls, the cytoskeleton determines the shape of the cell. This is one of the functions of the intermediate filaments.

Cell movement. The dynamic collection of microfilaments and microtubles can be continually in the process of assembly and disassembly, resulting in forces that move the cell. There can also be sliding motions of these structures. Audesirk and Audesirk give examples of white blood cells “crawling” and the migration and shape changes of cells during the development of multicellular organisms.

Organelle movement. Microtubules and microfilaments can help move organelles from place to place in the cell. In endocytosis a vesicle formed engulfs a particle abutting the cell. Microfilaments then attach to the vesicle and pull it into the cell. Much of the complex synthesis and distribution function of the endoplasmic reticulum and the Golgi complex makes use of transport vescicles,  associated with the cytoskeleton.

Cell division. During cell division, microtubules accomplish the movement of the chromosones to the daughter nucleus. Also, a ring of microfilaments helps divide two developing cells by constricting the central region between the cells (fission).

Hickman, et al. Ch 4 Hickman, Cleveland P., Roberts, Larry S., and Larson, Allan, Integrated Principles of Zoology, 9th. Ed., Wm C. Brown, 1995.
Audesirk & Audesirk Ch 6 Audesirk, Teresa and Audesirk, Gerald, Biology, Life on Earth, 5th Ed., Prentice-Hall, 1999.

Intermediate filaments are 8-12 nanometers in diameter and are twisted together in a cord shape. They are composed of keratin and keratin-like proteins.  These filaments are tough and resist tension.

Microtubules are composed of alpha and beta tubulin that form long, hollow cylinders.  These are fairly strong proteins and are the largest component of cytoskeleton at 25 nanometers. Tubular monomers can be lengthened or shortened from the positive end.

Microtubules have three different functions.

They make up the cell’s

  1. centriole
  2. the flagella and cilia of a cell, and
  3. they serve as “tracks” for transport vesicles to move along.

Key Points 


  1. help the cell resist compression,
  2. provide a track along which vesicles can move throughout the cell, and
  3. are the components of cilia and flagella.

Cilia and flagella are hair-like structures that

  1. assist with locomotion in some cells, as well as
  2. line various structures to trap particles.

The structures of cilia and flagella are a “9+2 array,” meaning that

  • a ring of nine microtubules is surrounded by two more microtubules.

Microtubules attach to replicated chromosomes

  • during cell division and
  • pull them apart to opposite ends of the pole,
  • allowing the cell to divide with a complete set of chromosomes in each daughter cell.

Microtubules are the largest element of the cytoskeleton.

The walls of the microtubule are made of

  • polymerized dimers of α-tubulin and β-tubulin, two globular proteins.

With a diameter of about 25 nm, microtubules are the widest components of the cytoskeleton.

They help the cell

  • resist compression,
  • provide a track along which vesicles move through the cell, and
  • pull replicated chromosomes to opposite ends of a dividing cell.

Like microfilaments, microtubules can dissolve and reform quickly.

Microtubules are also the structural elements of flagella, cilia, and centrioles (the latter are the two perpendicular bodies of the centrosome). In animal cells, the centrosome is the microtubule-organizing center. In eukaryotic cells, flagella and cilia are quite different structurally from their counterparts in prokaryotes.

Intermediate Filaments

Intermediate filaments (IFs) are cytoskeletal components found in animal cells. They are composed of a family of related proteins sharing common structural and sequence features.

epithelial cells

epithelial cells

flagella and cilia share a common structural arrangement of microtubules called a “9 + 2 array.” This is an appropriate name because a single flagellum or cilium is made of a ring of nine microtubule doublets surrounding a single microtubule doublet in the center.

9+2 array

9+2 array

The `Spectraplakins’: cytoskeletal giants with characteristics of both spectrin and plakin families

Katja Röper, Stephen L. Gregory and Nicholas H. Brown
J Cell Sci Nov 15, 2002; 115: 4215-4225​jcs.00157





The sequential endosymbiotic origins of eukaryotes: Compared to bacteria and archaea, the typical eukaryotic cell is much more structurally complex.

While the prokaryotes have a rigid cell wall, the ancestral eukaryote appears to have been wall-less (the walls of plant cells appear to represent a adaptation, and are not homologous to prokaryotic cell walls).

In addition to a nucleus (wherein the cell’s DNA is located, and which we will return to in the next section), there are cytoskeletal structures, including distinctive flagella (quite different from those found in prokaryotes), an active (motile) plasma membrane, capable of engulfing other cells, and multiple internal membrane systems. (A more complete description of cell structure is beyond this version of Biofundamentals).

In aerobic bacteria and cyanobacteria, the electron transport chains associated with ATP synthesis (through either photosynthesis or aerobic respiration) located within the plasma membrane (and in the case of cyanobacteria, internal membrane systems as well).

The same processes (aerobic respiration and photosynthesis) occur within eukaryotic cells. Animals have aerobic respiration, while plants have both).

However, these processes do not occur on the plasma membrane, but rather within distinct cytoplasmic organelles: mitochondria for aerobic respiration and chloroplasts for photosynthesis. All eukaryotic cells have mitochondria, plants (which are eukaryotic) have both mitochondria and chloroplasts.

An intriguing evolutionary question was, are these processes related, that is, are the processes of aerobic respiration and photosynthesis found in eukaryotes homologous to the processes found in bacteria and cyanobacteria, or did they originate independently.

The path to understanding that homologous nature of these processes began with studies of cell structure.

spectrin protein superfamily.large

spectrin protein superfamily.large

The role of secreted factors and extracellular matrix

The role of secreted factors and extracellular matrix

Focal Adhesions: Transmembrane Junctions Between the Extracellular Matrix and the Cytoskeleton

K Burridge, K Fath, T Kelly, G Nuckolls, and C Turner
Ann Rev Cell Biol Nov 1988; 4: 487-525

the extracellular matrix (ECM) is a collection of extracellular molecules secreted by cells that

  • provides structural and biochemical support to the surrounding cells.[1]

Because multicellularity evolved independently in different multicellular lineages, the composition of ECM varies between multicellular structures; however,

  • cell adhesion,
  • cell-to-cell communication and
  • differentiation

are common functions of the ECM.[2]

The animal extracellular matrix includes

  • the interstitial matrix and
  • the basement membrane.[3]

Interstitial matrix is present between various animal cells (i.e., in the intercellular spaces).

Gels of polysaccharides and fibrous proteins

  • fill the interstitial space and act as
  • a compression buffer against the stress placed on the ECM.[4]

Basement membranes are sheet-like depositions of ECM on which various epithelial cells rest.

The Extracellular Matrix (ECM)

Mechanical support to tissues

Organization of cells into tissues

  1. Activation of signaling pathways (cell growth, proliferation; development); examples:
  2. TGF-β, integrins
  3. specialized roles (tendon, bone; cartilage; cell movement during development; basal lamina in epithelia)


  1. proteoglycans
  2. collagen fibers (mechanical strength)
  3. multiadhesive matrix proteins (linking cell surface receptors to the (ECM)

Integrin connects the extracellular matrix with the actin cytoskeleton inside the cell

Fibronectin Integrin

Fibronectin Integrin

Continuous membrane-cytoskeleton adhesion requires continuous accommodation to lipid and cytoskeleton dynamics.

Sheetz MP, Sable JE, Döbereiner HG.
Annu Rev Biophys Struct Biomol. 2006;35:417-34.

The plasma membrane of most animal cells conforms to the cytoskeleton and only occasionally separates to form blebs. Previous studies indicated that

  • many weak interactions between cytoskeleton and the lipid bilayer
  • kept the surfaces together to counteract the normal outward pressure of cytoplasm.

Either the loss of adhesion strength or the formation of gaps in the cytoskeleton enables the pressure to form blebs. Membrane-associated cytoskeleton proteins, such as spectrin and filamin, can

  • control the movement and aggregation of membrane proteins and lipids,
    e.g., phosphoinositol phospholipids (PIPs), as well as blebbing.

At the same time, lipids (particularly PIPs) and membrane proteins affect

  • cytoskeleton and signaling dynamics.

We consider here the roles of the major phosphatidylinositol-4,5-diphosphate (PIP2) binding protein, MARCKS, and PIP2 levels in controlling cytoskeleton dynamics. Further understanding of dynamics will provide important clues about how membrane-cytoskeleton adhesion rapidly adjusts to cytoskeleton and membrane dynamics.

Interaction of membrane/lipid rafts with the cytoskeleton: impact on signaling and function: membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling.

Head BP, Patel HH, Insel PA   Epub 2013 Jul 27.
Biochim Biophys Acta. 2014 Feb;1838(2):532-45.

The plasma membrane in eukaryotic cells contains microdomains that are

  • enriched in certain glycosphingolipids, gangliosides, and sterols (such as cholesterol) to form membrane/lipid rafts (MLR).

These regions exist as caveolae, morphologically observable flask-like invaginations, or as a less easily detectable planar form. MLR are scaffolds for many molecular entities, including

  • signaling receptors and ion channels that
  • communicate extracellular stimuli to the intracellular milieu.

Much evidence indicates that this organization and/or the clustering of MLR into more active signaling platforms

  • depends upon interactions with and dynamic rearrangement of the cytoskeleton.

Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to MLR and help

  • regulate lateral diffusion of membrane proteins and lipids in response to extracellular events
    (e.g., receptor activation, shear stress, electrical conductance, and nutrient demand).

MLR regulate

  • cellular polarity,
  • adherence to the extracellular matrix,
  • signaling events (including ones that affect growth and migration), and
  • are sites of cellular entry of certain pathogens, toxins and nanoparticles.

The dynamic interaction between MLR and the underlying cytoskeleton thus regulates many facets of the function of eukaryotic cells and their adaptation to changing environments. Here, we review general features of MLR and caveolae and their role in several aspects of cellular function, including

  • polarity of endothelial and epithelial cells,
  • cell migration,
  • mechanotransduction,
  • lymphocyte activation,
  • neuronal growth and signaling, and
  • a variety of disease settings.

This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Cell control by membrane–cytoskeleton adhesion

Michael P. Sheetz
Nature Reviews Molecular Cell Biology 2, 392-396 (May 2001) | http://dx.doi.doi:/10.1038/35073095

The rates of mechanochemical processes, such as endocytosis, membrane extension and membrane resealing after cell wounding, are known to be controlled biochemically, through interaction with regulatory proteins. Here, I propose that these rates are also controlled physically, through an apparently continuous adhesion between plasma membrane lipids and cytoskeletal proteins.

Lipid Rafts, Signalling and the Cytoskeleton

Lipid rafts are specialised membrane domains enriched in certain lipids cholesterol and proteins. The existence of lipid rafts was first hypothesised in 1988 (Simons & van Meer, 1988; Simon & Ikonen, 1997), but what we know as “caveolae” were first observed  much earlier (Palade, 1953; Yamada, 1955).  Caveolae are flask shaped invaginations on the cell surface that are a type of membrane raft, these were named “caveolae intracellulare” (Yamada, 1955).  After a long argument (Jacobson & Dietrich, 1999), most now consider that these rafts actually exist, however, there is some confusion surrounding the classification of these rafts. It presently seems that there could be three types; caveolae, glycosphingolipid enriched membranes (GEM), and polyphospho inositol rich rafts. It may also be that there are inside rafts (PIP2 rich and caveolae) and outside rafts (GEM).

The fatty-acid chains of lipids within the rafts tend to be extended and so more tightly packed, creating domains with higher order. It is therefore thought that  rafts exist in a separate ordered phase that floats in a sea of poorly ordered lipids.  Glycosphingolipids, and other lipids with long, straight acyl chains are preferentially incorporated into the rafts.

Caveolae are similar in composition to GEMs that lack caveolae and in fact cells that lack caveolin-1 do not have morphologically identifiable caveolae but instead have extra GEM.  These cells can then be transfected with caveolin-1 cDNA and the caveolae then appear.  This suggests that GEM are merely caveolae without caveolin-1.  Caveolin-1 is a 21kDa integral membrane protein that binds cholesterol (Maruta et al, 1995). In cells lacking caveolin-1, caveolin-2 is synthesised but remains in the Golgi.  Caveolin 1 and 2 colocalise when expressed in the same cells and they may form hetero-dimers (Scherer et al, 1997). Caveolin-3 is expressed in muscle where it forms muscle-type caveolae.  Caveolin-3 is involved in certain types of muscular dystrophy (Galbiati et al, ). A slightly confusing finding is that caveolae are the reported site of integrin signalling ().  It is difficult to imagine integrins being available in the depths of membrane invaginations for binding extra-cellular ligands.

The function of rafts

Many functions have been attributed to rafts, from cholesterol transport, endocytosis and signal transduction.  The later is almost certainly the case. It has been suggested that the primary function of caveolae was in constitutive endocytic trafficking but recent data show that this is not the case, instead caveolae are very stable regions of membranes that are not involved in  endocytosis (Thompsen et al, 2002).

lipid raft

lipid raft

Rafts and the Cytoskeleton

Many actin binding proteins are known to bind to polyphosphoinositides and to be regulated by them (see PI and ABPs), by a series of protein domains such as PH, PX and ENTH (see Domains).  It is consequently scarcely surprising that some ABPs are suggested to link the actin cytoskeleton and PIP2-enriched rafts. One of these is gelsolin, a Ca2+, pH and polyphosphoinositide regulated actin capping and severing protein (see Gelsolin Family), that partitions into rafts isolated biochemically from brain (Fanatsu et al, 2000).

GEMs too are suggested to link to the actin cytoskeleton through ABPs particularly ERM proteins through EBP50, a protein that binds members of the ERM proteins through the ERM C-terminus (Brdickova et al, 2001).


Brdickova, N., Brdicka, T., Andrea, L., Spicka, J., Angelisova, P., Milgram, S. L. & Horejsi, V. (2001) Interaction between two adaptor proteins, PAG and EBP50: a possible link between membrane rafts and actin cytoskeleton.  FEBS letters. 507, 133-136.

Cary, L. A. & Cooper, J. A. (2000) Molecular switches in lipid rafts.  Nature. 404, 945-947.

Czarny, M., Fiucci, G., Lavie, Y., Banno, Y., Nozawa, Y. & Liscovitch, M. (2000) Phospholipase D2: functional interaction with caveolin in low-density membrane microdomains.,  FEBS letters.

Foger, N., Funatsu, N., Kumanogoh, H., Sokawa, Y. & Maekawa, S. (2000) Identification of gelsolin as an actin regulatory component in a Triton insoluble low density fraction (raft) of newborn bovine brain.  Neuroscience Research. 36, 311-317.

Galbiati, F., Engelman, J. A., Volonte, D., Zhang, X. L., Minetti, C., Li, M., Hou jr, H., Kneitz, B., Edelman, W. & Lisanti, M. P. (2001) Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and T-tubule abnormalities.  J. Biol.Chem. 276, 21425-21433.

…  (more)

centralpore-small  Gating and Ion Conductivity

centralpore-small Gating and Ion Conductivity

Interaction of epithelial ion channels with the actin-based cytoskeleton.

Mazzochi C, Benos DJ, Smith PR.
Am J Physiol Renal Physiol. 2006 Dec;291(6):F1113-22. Epub 2006 Aug 22

The interaction of ion channels with the actin-based cytoskeleton in epithelial cells

  • not only maintains the polarized expression of ion channels within specific membrane domains,
  • it also functions in the intracellular trafficking and regulation of channel activity.

Initial evidence supporting an interaction between

  • epithelial ion channels and the
  • actin-based cytoskeleton

came from patch-clamp studies of the effects of cytochalasins on channel activity. Cytochalasins were shown to

  • either activate or inactivate epithelial ion channels.

An interaction between

  • the actin-based cytoskeleton and epithelial ion channels

was further supported by the fact that the addition of monomeric or filamentous actin to excised patches had an effect on channel activity comparable to that of cytochalasins. Through the recent application of molecular and proteomic approaches, we now know that

  • the interactions between epithelial ion channels and actin can either be direct or indirect,
  • the latter being mediated through scaffolding or actin-binding proteins
  • that serve as links between the channels and the actin-based cytoskeleton.

This review discusses recent advances in our understanding of the interactions between epithelial ion channels and the actin-based cytoskeleton, and the roles these interactions play in regulating the cell surface expression, activity, and intracellular trafficking of epithelial ion channels.

epithelial ion channels

epithelial ion channels

Actin cytoskeleton regulates ion channel activity in retinal neurons.

Maguire G, Connaughton V, Prat AG, Jackson GR Jr, Cantiello HF.
Neuroreport. 1998 Mar 9;9(4):665-70

The actin cytoskeleton is an important contributor to the integrity of cellular shape and responses in neurons. However, the molecular mechanisms associated with functional interactions between the actin cytoskeleton and neuronal ion channels are largely unknown. Whole-cell and single channel recording techniques were thus applied to identified retinal bipolar neurons of the tiger salamander (Ambystoma tigrinum) to assess the role of acute changes in actin-based cytoskeleton dynamics in the regulation of voltage-gated ion channels. Disruption of endogenous actin filaments after brief treatment (20-30 min) with cytochalasin D (CD) activated voltage-gated K+ currents in bipolar cells, which were largely prevented by intracellular perfusion with the actin filament-stabilizer agent, phalloidin. Either CD treatment under cell-attached conditions or direct addition of actin to excised, inside-out patches of bipolar cells activated and/or increased single K+ channels. Thus, acute changes in actin-based cytoskeleton dynamics regulate voltage-gated ion channel activity in bipolar cells.

Cytoskeletal Basis of Ion Channel Function in Cardiac Muscle

Matteo Vatta, Ph.D1,2 and Georgine Faulkner, Ph.D3

The publisher’s final edited version of this article is available at Future Cardiol

The heart is a force-generating organ that responds to self-generated electrical stimuli from specialized cardiomyocytes. This function is modulated by sympathetic and parasympathetic activity.

In order to contract and accommodate the repetitive morphological changes induced by the cardiac cycle,

  • cardiomyocytes depend on their highly evolved and specialized cytoskeletal apparatus.

Defects in components of the cytoskeleton, in the long term, affect

  • the ability of the cell to compensate at
  • both functional and structural levels.

In addition to the structural remodeling, the myocardium becomes

  • increasingly susceptible to altered electrical activity leading to arrhythmogenesis.

The development of arrhythmias secondary to structural remodeling defects has been noted, although the detailed molecular mechanisms are still elusive. Here I will review the current knowledge of the molecular and functional relationships between the cytoskeleton and ion channels and, I will discuss the future impact of new data on molecular cardiology research and clinical practice. 

Stretch-activated ion channel

Stretch-activated or stretch-gated ion channels are

  • ion channels which open their pores in response to
  • mechanical deformation of a neuron’s plasma membrane.

[Also see mechanosensitive ion channels and mechanosensitive channels, with which they may be synonymous]. Opening of the ion channels

  • depolarizes the afferent neuron producing an action potential with sufficient depolarization.[1]

Channels open in response to two different mechanisms: the prokaryotic model and the mammalian hair cell model.[2][3] Stretch-activated ion channels have been shown to detect vibration, pressure, stretch, touch, sounds, tastes, smell, heat, volume, and vision.[4][5][6] Stretch-activated ion channels have been categorized into

three distinct “superfamilies”:

  1. the ENaC/DEG family,
  2. the TRP family, and
  3. the K1 selective family.

These channels are involved with bodily functions such as blood pressure regulation.[7] They are shown to be associated with many cardiovascular diseases.[3] Stretch-activated channels were first observed in chick skeletal muscles by Falguni Guharay and Frederick Sachs in 1983 and the results were published in 1984.[8] Since then stretch-activated channels have been found in cells from bacteria to humans as well as plants.

Mechanosensitivity of cell membranes. Ion channels, lipid matrix and cytoskeleton.

Petrov AG, Usherwood PN.
Eur Biophys J. 1994;23(1):1-19

Physical and biophysical mechanisms of mechano-sensitivity of cell membranes are reviewed. The possible roles of

  • the lipid matrix and of
  • the cytoskeleton in membrane mechanoreception

are discussed. Techniques for generation of static strains and dynamic curvatures of membrane patches are considered. A unified model for

  • stress-activated and stress-inactivated ion channels

under static strains is described. A review of work on

  • stress-sensitive pores in lipid-peptide model membranes

is presented. The possible role of flexoelectricity in mechano-electric transduction, e.g. in auditory receptors is discussed. Studies of

  • flexoelectricity in model lipid membranes, lipid-peptide membranes and natural membranes containing ion channels

are reviewed. Finally, possible applications in molecular electronics of mechanosensors employing some of the recognized principles of mechano-electric transduction in natural membranes are discussed.Marhaba, R. & Zoller, M. (2001) Involvement of CD44 in cytoskeleton rearrangement and raft reorganization in T cells.  J.Cell Sci. 114, 1169-1178.

FIGURE 2 | The transient pore model.

peroxisomal matrix protein

peroxisomal matrix protein

Peroxisomal matrix protein import: the transient pore model

Ralf Erdmann & Wolfgang Schliebs
Nature Reviews Molecular Cell Biology 6, 738-742 (September 2005)

Peroxisomal matrix protein import: the transient pore model
The transient pore model

The peroxisomal import receptor peroxin-5 (Pex5) recognizes peroxisomal targeting signal-1 (PTS1)-containing cargo proteins in the cytosol. It then moves to the peroxisome where it inserts into the peroxisomal membrane to become an integral part of the protein-import apparatus. Pex14 and/or Pex13, which are associated with Pex17, are proposed to be involved in tethering the receptor to the membrane and in the assembly, stabilization and rearrangement of the translocon. Cargo release into the peroxisomal matrix is thought to be initiated by intraperoxisomal factors — for example, the competitive binding of the intraperoxisomal Pex8, which also has a PTS1. The disassembly and recycling of Pex5 is triggered by a cascade of protein–protein interactions at the peroxisomal membrane that results in the Pex1-, Pex6-driven, ATP-dependent dislocation of Pex5 from the peroxisomal membrane to the cytosol. Pex1 and Pex6 are AAA+ (ATPases associated with a variety of cellular activities) peroxins that are associated with the peroxisome membrane through Pex15 in yeast or its orthologue PEX26 in mammals. Pex4, which is membrane-anchored through Pex22, is a member of the E2 family of ubiquitin-conjugating enzymes, and Pex2, Pex10 and Pex12 contain the RING-finger motif that is a characteristic element of E3 ubiquitin ligases. Mono- or di-ubiquitylation are reversible steps that seem to be required for the efficient recycling of import receptors, whereas polyubiquitylation might signal the proteasome-dependent degradation of receptors when the physiological dislocation of receptors is blocked. Ub, ubiquitin.

Nature Reviews Molecular Cell Biology 6, 738-742 (September 2005) |


peroxisomal protein pore model

peroxisomal protein pore model

Peroxisomal matrix protein import: the transient pore model

Ralf Erdmann & Wolfgang Schliebs
Nature Reviews Molecular Cell Biology 6, 738-742 (September 2005)

Peroxisomal matrix protein import: the transient pore model

Peroxin-13 (Pex13), Pex14 and Pex17 are constituents of the docking complex for cycling peroxisomal import receptors. Another protein assembly in the peroxisomal membrane comprises the RING-finger-motif-containing peroxins Pex2, Pex10 and Pex12. This motif is a characteristic element of E3 ubiquitin ligases, and this subcomplex is linked to the docking complex by Pex8, which is peripherally attached to the lumenal side of the peroxisomal membrane. Pex4 is a member of the E2 family of ubiquitin-conjugating enzymes and is anchored to the peroxisomal membrane through the cytosolic domain of Pex22. Pex1 and Pex6 are interacting AAA+ proteins (ATPases associated with a variety of cellular activities), which are attached to the membrane through binding to Pex15 in yeast or to its mammalian counterpart PEX26.

Peroxisomal matrix protein import: the transient pore model

Ralf Erdmann & Wolfgang Schliebs

Peroxisomes import folded, even oligomeric, proteins, which distinguishes the peroxisomal translocation machinery from the well-characterized translocons of other organelles. How proteins are transported across the peroxisomal membrane is unclear. Here, we propose a mechanistic model that conceptually divides the import process into three consecutive steps: the formation of a

  • translocation pore by the import receptor,
  • the ubiquitylation of the import receptors, and
  • pore disassembly/receptor recycling.


Masoud Naderi Maralani

Identification of the phytosphingosine metabolic pathway leading to odd-numbered fatty acids

The long-chain base ​phytosphingosine is a component of sphingolipids and exists in yeast, plants and some mammalian tissues. ​Phytosphingosine is unique in that it possesses an additional hydroxyl group compared with other long-chain bases. However, its metabolism is unknown. Here we show that ​phytosphingosine is metabolized to odd-numbered fatty acids and is incorporated into glycerophospholipids both in yeast and mammalian cells. Disruption of the yeast gene encoding long-chain base 1-phosphate lyase, which catalyzes the committed step in the metabolism of ​phytosphingosine to glycerophospholipids, causes an ~40% reduction in the level of phosphatidylcholines that contain a C15 fatty acid. We also find that ​2-hydroxypalmitic acid is an intermediate of the phytosphingosine metabolic pathway. Furthermore, we show that the yeast ​MPO1 gene, whose product belongs to a large, conserved protein family of unknown function, is involved in ​phytosphingosine metabolism. Our findings provide insights into fatty acid diversity and identify a pathway by which hydroxyl group-containing lipids are metabolized.

About GPCRs

G-protein-coupled receptors (GPCRs) are a class of membrane proteins that allow the transmission of a wide variety of signals over the cell membrane, between different cells and over long distances inside the body. The molecular mechanisms of action of GPCRs were worked in great detail by Brian Kobilka and Robert Lefkowitz for which they were jointly awarded the Nobel Prize in Chemistry for 2012. Read More

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Mechanisms of Drug Resistance

Curator: Larry H. Bernstein, MD, FCAP

Leaders in Pharmaceutical Intelligence, CSO


Mechanisms of Drug Resistance

This discussion is a continuing discussion of matters of metabolomics and the
essential role of genomic or epigenetic mechanisms to guide the development of
proteomic driven effectors of resistance to drug therapy.
We start with the elucidation of efflux pumps in bacteria, and we conclude with
consideration of cancer cells.

Part 1. Antimicrobial Resistance

Antimicrobial resistance is the ability of microbes, such as bacteria, viruses,
parasites, or
fungi, to grow in the presence of a chemical (drug) that would normally kill it
or limit its growth.

difference between non-resistant bacteria and drug resistant bacteria

difference between non-resistant bacteria and drug resistant bacteria

Non-resistant bacteria multiply, and upon drug treatment, the bacteria die. Drug
resistant bacteria multiply as well, but upon drug treatment, the bacteria continue
to spread.

Many infectious diseases are increasingly difficult to treat because of antimicrobial-resistant organisms, including HIV infection, staphylococcal infection, tuberculosis,
influenza, gonorrhea, candida infection, and malaria.

Between 5 and 10 percent of all hospital patients develop an infection. About 90,000
of these patients die each year as a result of their infection, up from 13,300 patient
deaths in 1992.

According to the Centers for Disease Control and Prevention (April 2011), antibiotic
resistance in the United States costs an estimated $20 billion a year in excess health
care costs. In addition, a cost of $35 million in other societal costs and more than 8
million additional days that people spend in the hospital. This is because people
infected with antimicrobial-resistant organisms are more likely to have longer hospital stays and may require more complicated treatment.

Diagnostic tests designed to determine which microbe is causing infection and to
which antimicrobials the microbe might be resistant take a few days or weeks to give
results because of a requirement for the microbe to grow for it to be identified.

Part 2. Antibiotic Tolerance   
Reported By Jef Akst | June 25, 2014

Optimization of lag time underlies antibiotic tolerance in evolved bacterial

O. Fridma et al.    Nature, 2014

Populations of Escherichia coli grown in the lab develop tolerance when exposed to
repeated treatments with the antibiotic ampicillin. The bacteria evolved to stay in a
dormant “lag” phase for just longer than three-, five-, or eight-hour-long treatment
courses. Antibiotic tolerance, which allows bacteria to survive even high levels of
antibiotics by remaining dormant. Tolerance may lead to an inaccurate assumption
that an unsuccessful antibiotic treatment failed as a result of resistance, in which
the microbe has evolved to grow in the presence of the drug. Resistance is very well
known; but the issue of tolerance is much less known,” according to Tom Coenye of
the Laboratory of Pharmaceutical Microbiology (LPM) at Gent University in Belgium,
who was not involved in the research.  This is a new phenomenon, extended lag,
where mutants have a longer lag time, and that extended lag allows them to survive
an attack by antibiotics.

To gain a better understanding of how bacterial populations might evolve to tolerate
antibiotic exposure, Nathalie Q. Balaban, a microbiologist and physicist at The Hebrew
University of Jerusalem in Israel and her colleagues exposed cultures of E. coli to high
concentrations of ampicillin for three, five, or eight hours, then washed the drug away
and suspended the bacteria in fresh media to be grown overnight. The next day, the
team repeated these treatments. In 10 cycles we could see that tolerance had evolved,
” Balaban said. Indeed, while the ampicillin treatments killed more than 99.9 percent of
the E. coli, by day 10, bacterial survival had increased 100-fold.

Moreover, the bacteria were also tolerant to norfloxacin, an antibiotic with a different mechanism of action than ampicillin but also ineffective during the dormant stage,
further supporting the idea that the E. coli populations had evolved to tolerate certain
durations of antibiotic exposure. “This is characteristic of tolerance,” said Balaban.
“The bacteria that have evolved tolerance under ampicillin are also more tolerant to
this completely different class of antibiotics.” Resistance, on the other hand, is usually
class-specific, she noted.

The researchers identified three genes that seemed to play a functional role in antibiotic
tolerance. While the exact mechanism of how mutations in these genes may have
lengthened the bacteria’s lag time is not yet known, two of the genes are part of pathways
that were previously implicated in bacterial persistence, including an antitoxin in a
common toxin-antitoxin module
 that may help regulate that bacteria’s growth.

Part 3. Multidrug Resistance Perspective

Mechanisms of antibiotic resistance in salmonella: efflux pumps, genetics,
quorum sensing and biofilm formation.

Perspectives in Drug Discovery and Design 02/2011; 8:114-123.
Martins M, McCusker, Amaral, Fanning S

Multidrug resistance (MDR) to antibiotics presents a serious therapeutic problem
in the treatment of bacterial infections. The importance of this mechanism of resistance
in clinical settings is reflected in the increasing number of reports of multidrug resistant
isolates. In Salmonella enterica, the most common etiological agent of food borne
salmonellosis worldwide, MDR is becoming a major concern.

In Salmonella the main mechanisms of antibiotic resistance are mutations in target
genes (such as DNA gyrase and topoisomerase IV) and the over-expression of efflux pumps. However, other mechanisms such as

  1. changes in the cell envelope;
  2. down regulation of membrane porins;
  3. increased lipopolysaccharide (LPS) component of the outer cell membrane;
  4. quorum sensing and
  5. biofilm formation

can also contribute to the resistance seen in this microorganism. To overcome
this problem new therapeutic approaches are urgently needed.

In the case of efflux-mediated multidrug resistant isolates, one of the treatment
options could be

  • the use of efflux pump inhibitors (EPIs)
  • in combination with the antibiotics to which the bacteria is resistant.

By blocking the efflux pumps

  • resistance is partly or wholly reversed,
  • allowing antibiotics showing no activity against the MDR strains
  • to be used to treat these infections.

Compounds that show potential as an EPI are therefore of interest, as well as new
strategies to target the efflux systems. Quorum sensing (QS) and biofilm formation
are systems also known to be involved in antibiotic resistance. Consequently,
compounds that

  • can disrupt or inhibit these bacterial “communication systems” will be of use in
    the treatment of these infections.

Part 5. Effux pumps and S. Aureus

Multidrug Efflux Pumps in Staphylococcus aureus: an Update

SS Costa, M Viveiros, L Amaral and I Couto
1Grupo de Micobactérias, Unidade de Microbiologia Médica, Instituto de Higiene e
Medicina Tropical, Universidade Nova de Lisboa (IHMT, UNL), 2Centro de Recursos
Microbiológicos (CREM), UNL, Portugal,3COST ACTION BM0701 (ATENS), Brussels,
The Open Microbiology Journal 2013;(Suppl 1-M5): 59-71

The emergence of infections caused by multi- or pan-resistant bacteria in the hospital
or in the community settings is an increasing health concern. Albeit there is no single
resistance mechanism behind multi-resistance, multidrug efflux pumps,

  • proteins that cells use to detoxify from noxious compounds,

seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria.
During the last decades, experimental data has established their contribution to low
level resistance to antimicrobials in bacteria and their

  • potential role in the appearance of MDR phenotypes, by the extrusion of multiple,
    unrelated compounds.

Recent studies suggest that

  • efflux pumps may be used by the cell as a first-line defense mechanism,

avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration
occurs, that allows survival in the presence of that agent.

In this paper we review the current knowledge on

  • MDR efflux pumps and their
  • intricate regulatory network in Staphylococcus aureus,

a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that

  • aureus MDR efflux pumps,
  • either chromosomal or plasmid-encoded, have
  • on resistance towards different antimicrobial agents and
  • on the selection of drug – resistant strains.

We will also discuss the many questions that still remain on the role of each specific
efflux pump and the need to establish appropriate methodological approaches to
address all these questions.

        Table 1. Multidrug Efflux Pumps Described for Staphylococcus aureus

Efflux Pump  Family Regulator(s) Substrate Specificity  References 
Chromosomally-encoded Efflux Systems 
NorA MFS MgrA,
Hydrophilic fluoroquinolones (ciprofloxacin,
norfloxacin) QACs (tetraphenylphosphonium,
benzalkonium chloride) Dyes (e.g. ethidium
bromide, rhodamine)
NorB MFS MgrA,
Fluoroquinolones (e.g. hydrophilic: ciprofloxacin,
norfloxacin and hydrophobic: moxifloxacin,
sparfloxacin) Tetracycline QACs (e.g.
tetraphenylphosphonium, cetrimide) Dyes (e.g. ethidium bromide)
NorC MFS MgrA(?),
Fluoroquinolones (e.g. hydrophilic: ciprofloxacin
and hydrophobic: moxifloxacin) Dyes
(e.g. rhodamine)
MepA MATE MepR Fluoroquinolones (e.g. hydrophilic: ciprofloxacin,
norfloxacin and hydrophobic: moxifloxacin,
sparfloxacin) Glycylcyclines (e.g. tigecycline) QACs (e.g. tetraphenylphosphonium, cetrimide, benzalkonium chloride) Dyes
(e.g. ethidium bromide)
MdeA MFS n.i. Hydrophilic fluoroquinolones (e.g. ciprofloxacin,
norfloxacin) Virginiamycin, novobiocin, mupirocin,
fusidic acid QACs (e.g. tetraphenylphosphonium,
benzalkonium chloride, dequalinium) Dyes (e.g. ethidium bromide)
SepA n.d. n.i. QACs (e.g. benzalkonium chloride) Biguanidines
(e.g. chlorhexidine) Dyes (e.g. acriflavine)
SdrM MFS n.i. Hydrophilic fluoroquinolones (e.g. norfloxacin) Dyes (e.g. ethidium bromide, acriflavine) [42]
LmrS MFS n.i. Oxazolidinone (linezolid) Phenicols
(e.g. choramphenicol, florfenicol) Trimethoprim, erythromycin, kanamycin,
fusidic acid QACs (e.g. tetrapheny-
lphosphonium) Detergents (e.g. sodium
docecyl sulphate) Dyes (e.g. ethidium
Plasmid-encoded Efflux Systems

QacA MFS QacR QACs (e.g. tetraphenylphosphonium,
benzalkonium chloride, dequalinium)
Biguanidines (e.g. chlorhexidine)
Diamidines (e.g. pentamidine) Dyes
(e.g. ethidium bromide,
rhodamine, acriflavine)
QacB MFS QacR QACs (e.g. tetraphenylphosphonium,
benzalkonium chloride)Dyes (e.g. ethidium bromide, rhodamine,
Smr SMR n.i. QACs (e.g. benzalkonium chloride,
cetrimide) Dyes (e.g. ethidium bromide)
QacG SMR n.i. QACs (e.g. benzalkonium chloride,
cetyltrymethylammonium) Dyes
(e.g. ethidium bromide)
QacH SMR n.i. QACs (e.g. benzalkonium chloride,
cetyltrymethylammonium) Dyes
(e.g. ethidium bromide)
QacJ SMR n.i. QACs (e.g. benzalkonium chloride,
cetyltrymethylammonium) Dyes
(e.g. ethidium bromide)

a n.d.: The family of transporters to which SepA belongs is not elucidated to date.
b n.i.: The transporter has no regulator identified to date.
QACs: quaternary ammonium compounds

Identification of the plasmid-encoded qacA efflux pump gene
in meticillin-resistant Staphylococcus aureus (MRSA)
strain HPV107, a representative of the MRSA Iberian clone

S.S. Costaa,b, E. Ntokouc, A. Martinsa,d, M. Viveirosa,e, S. Pournarasc,
I. Coutoa,b, L. Amarala,d,e,∗
a Unidade de Micobactérias, Instituto de Higiene e Medicina Tropical,
Universidade Nova de Lisboa (IHMT, UNL), b Centro de Recursos Microbiológicos,
Universidade Nova de Lisboa (CREM, UNL), d Unidade de Parasitologia e
Microbiologia Médica (UPMM), Instituto de Higiene e Medicina Tropical, Universidade
Nova de Lisboa (IHMT, UNL), Lisbon, Portugal; e COST ACTION BM0701 (ATENS)
c Department of Microbiology, Medical School, University of Thessaly, Larissa, Greece;
Int J Antimicrobial Agents  2010; 36: 557–561

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial
bacterium for which prevention and control measures consist mainly of

  • the application of biocides with antiseptic and disinfectant activity.

In this study, we demonstrated the presence of

  • the plasmid-located efflux pump gene qacA in MRSA strain HPV107,

a clinical isolate representative of the MRSA Iberian clone. The existence
of efflux activity in strain HPV107 due to the QacA pump was found and

  • this QacA efflux activity was linked with a phenotype of
  • reduced susceptibility towards several biocide compounds.

No association could be made with antibiotic resistance. This work
emphasises the potential of QacA pump activity in

  • the maintenance and dissemination of important MRSA strains in
    the hospital setting and, increasingly, in the community.

Efflux-mediated response of Staphylococcus aureus exposed to
ethidium bromide

I Couto1,2, S S Costa1, M Viveiros1, M Martins1,3 and L Amaral1,3*
1Unidade de Micobacterias, Instituto de Higiene e Medicina Tropical,
Universidade Nova de Lisboa (UNL), 2Centro de Recursos Microbiolo´gicos (CREM), Faculdade de Cieˆncias e Tecnologia, UNL,3UPMM,
Instituto de Higiene e Medicina Tropical, UNL, Portugal
J Antimicrob Chemother  (2008) 62, 504–513

By adapting an antibiotic-susceptible Staphylococcus aureus strain to
increasing concentrations of ethidium bromide, a known substrate
of efflux pumps (EPs), and

  • by phenotypically and genotypically analysing the resulting progeny,
  • we characterized the molecular mechanisms of S. aureus
    adaptation to ethidium bromide.

ATCC 25923 was grown in increasing concentrations of ethidium bromide.
The MICs of representatives of eight classes of antibiotics, eight biocides
and two dyes against ATCC 25923 and its ethidium bromide-resistant progeny
ATCC 25923EtBr were determined

  • with or without six efflux pump inhibitors (EPIs).

Efflux activity in the presence/absence of EPIs was evaluated by realtime
fluorometry. The presence and expression of eight EP genes were assayed
by PCR and quantitative RT–PCR (qRT–PCR), respectively. Mutations in
grlA, gyrA and norA promoter regions were screened by DNA sequencing.

Compared with its parental strain, ATCC 25923EtBr was

  • 32-fold more resistant to ethidium bromide and
  • also more resistant to biocides and hydrophilic fluoroquinolones.
  • Resistance to these could be reduced by the EPIs chlorpromazine,
    thioridazine and reserpine.

Increased efflux of ethidium bromide by ATCC 25923EtBr could be
inhibited by the same EPIs. qRT–PCR showed that

  • norA was 35-fold over-expressed in ATCC 25923EtBr,

whereas the remaining EP genes showed no significant increase in their

expression. Sequencing of the norA promoter region revealed

  • a 70 bp deletion in ATCC 25923EtBr.

Exposure of S. aureus to quaternary compounds such as ethidium bromide
results in decreased susceptibility of the organism to a wide variety of
compounds, including quinolones and biocides

  • through an efflux-mediated response, which
  • for strain ATCC 25923 is mainly NorA-mediated.

This altered expression may result from alterations in the norA
promoter region

Ethnic consumption of plant leaf extracts and appraisal of
their nutraceutical efficacy against multidrug resistant
staphylococcus aureus

Kaushik S1, 2*, Tomar Rs1, Shrivastav V1, Shrivastav A2 And Jain Sk3
Amity Institute of Biotechnology, Amity University Madhya Pradesh,
Gwalior (M.P.);  2: College of Life Sciences, Cancer Hospital and
Research Institute, Gwalior (M.P.); 3: Department of Microbiology,
Vikram University, Ujjain (M.P.), INDIA
IJBPAS, Feb, 2014, 3(2): 204-209

Nutraceuticals are natural bioactive chemical compounds that have
health promoting, disease preventing or medicinal properties.
Emergence of Multi Drug Resistant Staphylococci is increasing at
alarming rates and diseases caused by these strains leave patients
against multiple resistant Staphylococcus aureus.

The test bacteria were isolated and characterized by standard and
NCCLS recommended microbiological techniques. A total of eighteen
plant extracts were analysed for their antimicrobial activity. The
selection of medicinal plants was based on their traditional uses in
India. However most of these plants were not previously screened.
Antibacterial activity of these components was performed by standard
Kirby Bauer Disk Diffusion method approved by NCCLS and the
inhibitory effect was analysed by calculating Zone of inhibition.

Among the eighteen plant extracts analysed we found highest
activity in the effect of chemotherapy and as promising bio control agents

  • Guava,
  • Mango,
  • Jamun and
  • Pomengrate plant extracts,

while most of the other plants were either showing very moderate/
least activity against test bacteria. Our recent experiment indicated
that phytochemicals extracted with methanol can be utilized as
nutraceutical to lower the side.

Part 6. Efflux pumps and gram-negative organisms

Efflux Pumps that Bestow Multi-Drug Resistance of Pathogenic Gram-

Amaral L1,2*, Spengler G2, Martins A2,3 and Molnar J2
1Travel Medicine of the Centre for Malaria and Other Tropical Diseases (CMDT),
Institute of Hygiene and Tropical Medicine, Lisbon, Portugal 2Department of
Medical Microbiology and Immunobiology, Faculty of Medicine, University of
Szeged, Szeged, Hungary 3Unit of Parasitology and Medical Microbiology
(UPMM), Institute of Hygiene and Tropical Medicine, Lisbon, Portugal
Amaral et al., Biochem Pharmacol 2013; 2:3

The efflux pump

The efflux pump

Efflux pumps are integral plasma membrane protein systems that recognize and bind
noxious compounds present in the cytoplasm (toxic products produced by metabolism;
compounds that have penetrated the cell), or periplasm of the bacterial cell and extrude
it into the environment in which the bacterium resides [1].

The efflux pump machinery gives the cell additional protection to the one provided by

  • the constituents of its cell wall (example: lipopolysaccharides), and
  • provides an initial protection to noxious agents present in its
    natural environment that have penetrated into the cell (example: bile
    salts in the colon) [1].

The efflux pump machinery is divided into five superfamily classes;

  • the major facilitator (MF),
  • the ATP-binding cassette (ABC),
  • the resistance-nodulation-division (RND),
  • the small multi-drug resistance (SMR) and
  • the multi-drug and toxic compound extrusion (MATE).

With respect to Gram-negative bacteria, although they all play
important roles in the protection of the bacterium from noxious
agents present in the environment, the

  • main efflux pump of the Gram negative bacterium is a
    member of the RND superfamily, and
  • because multi-drug resistance of clinical isolates have
    been associated with the over-expression of this pump,

it has received a great deal of attention [2].

The first in vitro response of bacteria to a given noxious agent,
such as an antibiotic, is to over-express its main efflux pump [2].
If the bacterium is serially exposed in vitro to increasing
concentrations of that compound, it responds by increasing
the effective number of its main efflux pump, as well as others
that provide redundant protection [2].

However, if that “adapted” bacterium is now maintained at a
constant level of a noxious agent, the level of efflux pump
activity increases up to a maximum, followed by a gradual
return of efflux pump activity to its basal level. Concomitant
to this process, an accumulation of mutations of essential
proteins located in the plasma membrane (example penicillin
binding proteins), mutations 30 S component of the ribosome
and gyrase take place [3]. These events suggest that when
the organism is faced with an environment that contains a
constant toxic level of a compound, and the cost for
maintaining an energy consuming system, such as that
needed for the energy dependent efflux pump, is too
great a price to pay.

Therefore, in order to survive in this unchanging environment,
other mechanisms are activated. For example, activation of a
mutator master gene is thought to be an important step at this
level, which results in the mutation of genes that code for
essential proteins, reversing the over-expression of efflux-
pumps, but still conferring the bacterial resistant to the
environmental pressure via other mechanism(s), yet
to be understood [4,5].

During therapy, the level of resistance increases many fold
higher than that of the initial infecting strain. Hence, clinical
isolates from treated patients often show much higher levels
of antibiotic resistance than that of their wild type counterpart
(sometimes it can even present a 1000 fold increase) [6].
At this stage, resistance is usually related to the presence
of mutations, which reduces the survival of the resistant

  • once it is transferred to a noxious agent-free environment

that contains the competing wild type counterpart [3,4].

Depending upon when during therapy a clinical strain is isolated,
its resistance to two or more antibiotic classes (multi-drug
resistance (MDR)), may be due entirely to over-expressed
efflux pumps; to a mixture of over-expressed efflux pumps
and increasing accumulation of mutations; and only to mutations [3,4].

The degree of resistance can readily be determined with
methods that employ compounds known for their modulation
of efflux pump activity, such as

  • phenothiazines [7] or phenyl-arginine-betanaphthylamide
  • the latter which competes with the antibiotic as
    substrate of the efflux pump [8].

If in presence of such compounds,

  • the MDR bacterium is rendered fully susceptible
    to the antibiotic(s) to which it was initially resistant,
  • resistance is most likely due to its overexpressed
    efflux pump systems.
  • Contributions made by accumulated mutations
    render the organism less and less affected by the EPI.

This type of information is of great value to clinicians faced
with long-term therapy of a bacterial infection that
progresses to an MDR phenotype. It should be understood
that although the Gram-negative bacterium has essentially
one main efflux pump, such as

  • the AcrAB (Escherichia coli) or
  • the MexAB (Pseudomonas aeruginosa),

the deletion of the main efflux pump results in the over-
expression of one or more other RND efflux pumps,
such as is the case for deletion of the AcrAB, followed by

  • the over-expression of the AcrEF pump [2].

Redundancy of as many as nine RND efflux pumps [2],
provides additional protection to the organism.

The pumps belonging to the RND family form

  • a tripartite complex together with
  • the periplasmic proteins belonging to the
    membrane fusion-protein (MFP) family and
  • the outer membrane channels.

RND transporters consist of

  • a transporter protein that recognises and
    binds the noxious agent
    in the cytoplasm or periplasm and
  • transports it to the contiguous channel (TolC),
  • ending at the surface of the outer membrane.

The transporter is attached to the plasma membrane
by two or three fusion proteins, which are believed to assist the

  • extrusion of the substrate by peristaltic actions [9].

Although the actual structure of RND efflux pumps
in the cell envelop is not completely understood,

  • the structure of the transporter, TolC and fusion
    proteins are well established for major Gram-negative
    bacteria [10].

The PMF energy dependent efflux pump most likely needs the
passage of hydronium ions through its internal cavity,

  • for the release of the substrate that is
  • in turn ejected into the TolC channel via the
  • peristaltic action of the fusion proteins [11].

A low pH,

  • the concentration of hydronium ions at the surface of the cell
  • results in a pH difference of 2 or 3 pH units compared
    to that of the milieu,

the surface concentration of hydronium ions

  • provides the force for the mobility of hydronium ions
  • through porins leading to the acidification of the periplasm,
  • providing the low pH needed by the transporter
  • for the release of the substrate.

At high pH, these hydronium ions come from

  • hydrolysis of ATP by ATP synthase, and
  • are passed into the transporter, thereby
  • reducing its internal pH, so that
  • the release of the substrates can take place [11,12].

EPIs, such as the phenothiazines chlorpromazine or thioridazine,

  • exert their inhibition at pH above 6, and
  • are thought to affect hydrolysis of ATP
  • denying the efflux pump transporter hydronium ions needed

for release of the bound substrate [11,12].

The search for EPIs that are clinically useful continues, although

with respect to thioridazine, this old neuroleptic has been shown

  • to inhibit efflux pumps of pathogenic mycobacteria [13], and
  • has been successfully used to treat extensively drug resistant
    tuberculosis infections [14].

The regulation of the main efflux pump of Escherichia coli may
take place via   distinct pathways. The induced synthesis of the
transporter component of the AcrAB efflux pump, when the
organism is exposed in vitro to a noxious agent,

  1. involves the activation of the stress gene soxS,
  2. followed by the activation of the local regulator marA,
  3. then by the activation of the transporter gene acrB [8].

In the case of Salmonella spp. two component resistance
mechanisms, such as the PmrA/PmrB system, directly
activate the master efflux pump regulator ram A gene [15].
The activation of the PmrA/PmrB system takes place
readily when Salmonella spp. is phagocytosed due to
the acidic nature of the phagolysosome [15], as follows:

  1. PmrB is a sensor that self-phosphorylates, and
  2. then transfers the phosphate to PmrA.
  3. PmrA activates a nine gene operon, which
  4. codes for Lipid A introduced into the nascent
    lipopolysaccharide layer of the outer membrane.
  5. The increased presence of Lipid A renders the
    phagocytosed bacterium practically immune to
    everything, including the hydrolases of the
    phagolysososome [15].

Although some EPIs are in clinical trials, none have yet to
reach the marketplace,    mainly due to their common
toxicity against healthy mammalian cells, affecting
intrinsic mammalian efflux pumps, as for example
those of the blood brain barrier. Lastly, it should be
noted that compounds that inhibit the efflux pump
of bacteria also have the capacity to promote the
removal of plasmids that carry antibiotic resistant
genes [16,17].

  1. Nikaido H, Pages JM (2012) Broad-specificity efflux
    pumps and their role in multidrug resistance of Gram-
    negative bacteria. FEMSMicrobiol Rev 36: 340-363.
  2. Viveiros M, Jesus A, Brito M, Leandro C, Martins M,
    et al. (2005) Inducement and reversal of tetracycline
    resistance in Escherichia coli K-12 and expression of
    proton gradient-dependent multidrug efflux pump
    genes. Antimicrob Agents Chemother 49: 3578-3582.
  3. Martins A, Couto I, Aagaard L, Martins M, Viveiros M
    (2007) Prolonged exposure of methicillin-resistant
    Staphylococcus aureus (MRSA) COL strain to
    increasing concentrations of oxacillin results in a
    multidrug-resistant phenotype. Int J Antimicrob
    Agent 29: 302-305.
  4. Martins A, Spengler G, Molnar J, Amaral L (2012)
    Sequential responses of bacteria to noxious agents
    (antibiotics) leading to accumulation of mutations
    and permanent resistance. Biochem Pharmacol J
    Open Access 1: 7.

Inhibitors of efflux pumps of Gram-negative
bacteria inhibit Quorum Sensing

Leonard Amaral, Joseph Molnar
1 Grupo de Micobacterias, Unidade de Microbacterilogia,
Centro de Malaria e Doenças Tropicais (CMDT), Instituto de
Higiene e Medicina Tropical, Universidade Nova de Lisboa,
Lisbon, Portugal; 2 Cost Action BM0701 (ATENS) of the
European Commission/European Science Foundation;
3 Department of Medical Microbiology and Immunobiology,
University of Szeged, Szeged, Hungary
Open Journal of Pharmacology, 2012, 2-2

Quorum Sensing (QS) systems of bacteria consist of

  • a producer of the QS signal and the responder.

The generation of a QS signal provides the means by which
a population can behave in a concerted manner such as

  • swarming, swimming and secretion of biofilm, etc.

Because concerted bahaviour bestows protection to the bacterial
species, and hence factors involved in the severity of an infection
such as virulence are products of QS systems, compounds that
inhibit the QS system have significant clinical relevance. Recent
evidence suggests that

  • the secretion of QS signals takes place via
  • the efflux pump system of the producer of the signal.

Interestingly, compounds such as phenothiazines and
trifluoromethyl ketones (TFs)

  • that inhibit proton motive force (PMF) activities such
    as swarming and swimming also
  • inhibit the PMF dependent efflux pump systems of
    bacteria and their QS   systems.

This review discusses the relationship between the efflux
pump, the QS system and the compounds that affect both.
Lastly, suggestions are made regarding classes of compounds
that have been shown

  • to inhibit PMF dependent efflux pumps and the need
  • to evaluate them for QS inhibitory properties.

Keywords: Quorum Sensing, QS signal, acylated hydroxyl
lactone (AHL), efflux pumps, Proton Motive Force (PMF),
inhibitors of efflux pumps, inhibitors of QS systems,
phenothiazines, Trifluormethyl Ketones (TFs), plants
sources for QS inhibitors

Efflux pumps of bacteria provide protection from noxious
agents that are present in the environment in which they
exist. Noxious agents may be naturally occurring compounds
present in environments outside and within the human.

Because over-expressed efflux pumps render antibiotic
therapy problematic, an intense search for agents that
inhibit specific efflux pumps of specific bacteria has
been conducted during the past decade [9].

Communication between bacteria of the same strain
or species and between species contributes to their
survival [11-13]. Communication involves the secretion
of signals that invoke a specific response from the responder
[11-13]. This  communication process is termed Quorum
sensing (QS). When it takes place between strains of the
same species,

  • communication is directed towards the reduction
    of population growth and
  • reducing the possibility of exceeding the nutritional
    support of the environment

Other signals may involve a population response that involves

  • the secretion of bioactive molecules that inhibit the
    replication of a competing population species [14-16]
    or even kill [biocidins) [17-21] or
  • promote a swarming effect that recruits members
    of the same species to migrate  to a specific location [22-24]
    similar to swarming by insects subsequent to signals
    indicating site of food [example bees).
  • biofilm, encase the bacteria at distances from each other
    [25-29] and within the matrix of this biofilm are
    channels used for further communication [30].

Biofilms are produced in the wild, at sites such as surfaces
of rocks which maintain the bacterial population in situ [31]
and are also produced at sites of the human colonized by
infecting bacteria [32, 33].

Agents that inhibit the QS response of the infecting bacterium
are obviously important and hence, the search for such agents
that inhibit the QS system and biofilm formation has been in
effect for the past two decades [11-13].

There is a relationship between efflux pumps (EP), QS and
biofilm (BF) secretion which has come to the forefront only
recently [13]. Control of this relationship is critical for
successful therapy of MDR bacterial infections which have
become rather commonplace. It is the intent of this review
to identify agents which may serve to interfere with the
complex system of EP-QS-BF interaction.

Proton motive force (PMF) dependent transporters obtain
their energy for function from the proton motive force. The
proton motive force is the result of cellular metabolism which
yields protons that are not used for coupling with molecular
oxygen and which are exported to the surface of the cell [43-45]
where they are distributed and bound to components of
the protective lipopolysaccharide layer that covers the cell
and constitutes a part of the outer cell wall of Gram-negative
[46] and the cell wall of Gram positive bacteria [47].

The larger the concentration of protons (hydronium ions)
on the surface of the cell with respect to their lower
concentration on the medial side of the cytoplasmic
membrane creates an electrochemical gradient that
is termed the proton motive force (PMF) [48].

Because hydronium ions cannot penetrate the cell wall
or the membrane, they may re-enter the cell only
through channels such as porins in general [49, 50].
The movement of these hydromium ions from the
surface of the cell to the periplasm or cytoplasm is
predicated upon systems that use the PMF as source
of energy-namely the resistance nodulation division
(RND) family of transporters.

E. coli has a multiplicity of efflux pumps that may
exceed 30 in number [51]. However, the main
efflux pump of this organism is the AcrAB-TolC
efflux pump [52, 53] which when deleted, its
function is replaced by the AcrEF-TolC efflux
pump [51]. Both efflux pumps are members
of the resistance nodulation division family of
transporters [51] and consist of three proteins:

  1. The transporter AcrB coded by the gene acrB and
    is intimately attached to the  plasma membrane;
  2. Two fusion proteins AcrA coded by the gene acrA
    that flank the AcrB transporter and are thought
    to assist the movement of a substrate through
    the AcrB transporter [35]; and,
  3. TolC which is also part of other tri-unit efflux pumps
    of the organism [35], is contiguous with the AcrB
    transporter and provides a conduit for the extrusion
    of the substrate [38].

Although the means for the recognition of the substrate to
be extruded appears to involve a pocket within the transporter,
it appears to be

  • defined by a phenyalanine residue [54].

Nevertheless, studies employing fluorochromes recognised by
the AcrB transporter indicate that the binding and release of
the substrate are pH dependent [55].

  • At low pH the dissociation of the substrate is high and
  • at high pH it is very slow.

In a physiological environment of ca. pH 7, if the dissociation
of the substrate is slow or not at all, then the effectiveness of
the pump to extrude a noxious agent would be nullified.
However, since the pump functions at this pH, conditions that
result in the dissociation of the substrate needed for continuous
pump action must involve a

  • decrease of the pH of the internal cavity of the pump
    to which the substrate is bound.

It has been postulated that the lowering of the pH takes place
by the generation of hydronium ions from metabolism [6] which

  • pass from the cytoplasmic side of the plasma membrane
    through the transporter.

At lower pH, there is no need for the generation of metabolically
derived  hydronium ions since these ions can be

  • diverted by the PMF from the surface of the cell
    to the periplasm via porins.

Whether hydronium ions are to be generated from the
hydrolysis of ATP at high pH or used for the synthesis
of ATP at low pH is a special

  • function of ATP synthase [56-58].

Model of the AcrAB-TolC efflux pump of a Gram-
negative bacterium

AcrAB-TolC efflux pump of a Gram-negative bacterium

AcrAB-TolC efflux pump of a Gram-negative bacterium

Hypothesis. At near neutral pH, Hydronium ions from hydrolysis of ATP
by ATP synthase pass through the AcrB

transporter, reduce the pH to a point that causes the release of the
substrate. When the hydronium ions reach the surface of the cell they
are distributed over that surface and bind to lipopolysaccharides
and basic amino acids. When there is a need for hydronium ions for
activity of the efflux pump and the pH is lower than neutral, and
the hydrolysis of ATP is not favoured, hydronium ions from the
surface of cell via the PMF mobilize through the Aqua porins
and reach the transporter where they are pushed through
the transporter by the peristaltic action caused by the fusion
proteins. Substrates bound to the transporter dissociate
when the pH is reduced by the flow of hydronium ions and
are carried out by the flow of water.

Inhibitors of bacterial efflux pumps
Inhibitors of the QS of bacteria

Because phenothiazines inhibit many energy dependent systems
of bacteria such as motility [89, 90, 95], and these phenothiazines
also inhibit efflux pumps of bacteria [6, 7, 9, 41, 51, 73, 74, 76-83],
there seems to be a correlation between an active efflux pump
system and a functional QS system. That this assumption is correct,
recent evidence has been provided showing that the efflux pumps of
the AHL responding environmental Chromobacterium violaceum
(CV026) bacterium and that of E. coli are inhibited by the phenothiazine
thioridazine (TZ) [12]. Because TZ is known to inhibit genes that
regulate and code for efflux pumps of bacteria [41, 119, 120], it is
possible that the inhibition of the responding CV0126 bacterium to
AHLs [12] involves the inhibition of genes that code and regulate
the efflux pump of the responder which is assumed to recognise the
AHL signal as an noxious agent and hence would extrude it to the
environment [12]. The inhibition of an efflux pump should manifest
itself as an inhibitor of the QS component responsible for biofilm

Since the discovery of berberine a powerful inhibitor of bacterial
efflux pumps [159], plants have become sources of inhibitors of
efflux pumps [160-164]. Given that efflux pumps and the  QS of
bacteria have an intimate relationship as described in this review,
attention has been focused on plants for potential sources of inhibitors
of efflux pumps and QS systems. Essential oils from Columbian
plants have yielded a large number of compounds that inhibit the
QS system of responding bacteria such as

  1. limonene-carvone , the
  2. citral (geranial-neral) (isolated from Lippia alba),
  3. α-pinene (from Ocotea sp.),
  4. β-pinene (from Swinglea glutinosa),
  5. cineol (from Elettaria cardamomun),
  6. α-zingiberene (from Zingiber officinale) and
  7.  pulegone (from Minthostachys mollis) [165].

Several other essential oils, in particular were shown to present
promising inhibitory properties for the short chain AHL quorum
sensing (QS) system in Escherichia coli containing the biosensor

  •  plasmid pJBA132, in  particular Lippia alba.

Citral was the only  essential oil that presented some activity for
the long chain AHL QS system in Pseudomonas putida containing

  •  the plasmid pRK-C12 [165].

The essence of this review is to correlate the relationship of the
efflux pump system to the QS system of bacteria via the use of
compounds that inhibit both systems. Simply put, inhibitors of
the efflux pump system also, when studied, inhibit the QS system
as well. Because the PMF dependent efflux pump system of Gram-
negatives that is overexpressed is responsible for the multi-drug
phenotype of the bacterium, compounds that affect the PMF of
the bacterium are candidates that will inhibit the activity of the
pump. Consequently, this inhibition will inhibit the secretion of
biofilm, and because biofilm is a deterrent to the action of antibiotics,
compounds that affect the efflux pump system are promising
candidates for clinical evaluation.

Limiting and controlling carbapenem-resistant
Klebsiella pneumonia

L Saidel-Odes, A Borer.
1Infection Control and Hospital Epidemiology Unit, 2Infectious
Diseases Institute, Soroka University Medical Center and the
Faculty of Health Sciences, Ben-Gurion University of the Negev,
Beer-Sheva, Israel
Infection and Drug Resistance 2014:7 9–14

Carbapenem-resistant Klebsiella pneumoniae (CRKP)

  • is resistant to almost all antimicrobial agents,
  • is associated with substantial morbidity and mortality, and
  • poses a serious threat to public health.

The ongoing worldwide spread of this pathogen emphasizes the
need for immediate intervention. This article reviews the global
spread and risk factors for CRKP colonization/infection, and
provides an overview of the strategy to combat CRKP dissemination

Outbreaks of CRKP that have occurred around the world have
been associated with the plasmid-encoded carbapenemase
K. pneumoniae carbapenemase (KPC),

  • a carbapenem-hydrolyzing β-lactamase.19

CRKP isolates are resistant to almost all available antimicrobials
and are susceptible

  • only to polymyxins and tigecycline;
  • a minority to the few remaining aminoglycosides,
    though resistance to these agents is increasingly reported.20,21

Several investigators have evaluated predictors for CRKP colonization.
The following summarizes various studies.

  1. In a multivariate analysis, prior use of macrolides and
    any antibiotic exposure $14 days remained the only
    independent factors associated with CRKP bacteremia
  2. Nosocomial isolation of CRKP was strongly favored by the
    selection pressure of carbapenem. In this study, prior
    treatment with fluoroquinolones was associated with
    decreased risk for the emergence of CRKP.
  3. Previous use of carbapenem and cephalosporin
  4. Nursing home residency before hospital admission, bedridden
    status, and previous antibiotic therapy
  5. exposure to fluoroquinolones
  6. the recipient of antibiotics
  7. intensive care unit (ICU) stay, and
  8. Poor functional status,
  9. Independent predictors of subsequent carbapenem-
    resistant Enterobacteriaceae (CRE) infection were
  • admission to the ICU,
  • having a central venous  catheter,
  • receipt of antibiotics, and
  • diabetes mellitus

Schwaber et al and the Israeli CRE Working Group enforced the
Israel Ministry of Health guidelines mandating physical separation
of hospitalized carriers of CRE and dedicated staffing and appointed
a professional task force charged with containment.19 The monthly
incidence of nosocomial CRE was reduced from 55.5 to 11.7 cases
per 100,000 patient days within 15 months.

Part 7.  Tuberculosis

The Mechanism by which the Phenothiazine Thioridazine
Contributes to Cure Problematic Drug-Resistant Forms
of Pulmonary Tuberculosis: Recent Patents for “New Use”

L Amaral1*, A Martins2,3, G Spengler2, A Hunyadi4 and J Molnar2
Recent Patents on Anti-Infective Drug Discovery 2013; 8(3):000-000

At this moment, over half million patients suffer from multi-drug
resistant tuberculosis (MDR-TB) according to the data from the WHO.
A large majority is terminally ill with essentially incurable pulmonary
tuberculosis. This herein mini-review provides the experimental and
observational evidence that a specific phenothiazine,

  • thioridazine,

will contribute to cure any form of drug-resistant tuberculosis. This
antipsychotic agent is no longer under patent  protection for its
initial use. The reader is informed on the recent developments

  • in patenting this compound for “new use” with a special
  • emphasis on the aspects of drug-resistance.

Given that economic motivation can stimulate the use of this drug
as an antitubercular agent, future prospects are also discussed.

Thioridazine is not the only phenothiazine that has been recommended
for therapy of pulmonary tuberculosis. In general, many phenothiazines
have been implicated for antitubercular activity [62, 80-86]. Among
these are

  • trifluoperazine [87-94],
  • methdilazine [95, 96],
  • promazine [97, 98],
  • promethazine [97, 98],
  • fluphenazin [99],
  • propiomazine [100], and
  • the methylene blue related toluidine blue [101].

There are phenothiazine compounds derived from the parental
methylene blue for therapy of pathologies unrelated to tuberculosis
that also possess

  •  antitubercular [44, 48] and/or antimalarial properties [44].

Moreover, derivatives made from any of the phenothiazines that
have in vitro activity against Mycobacterium tuberculosis are also
active [61, 67, 102, 103], suggesting ample opportunities for
patenting of new analogs developed from known, active phenothiazines
with even less side effects than those of TZ, as recently suggested by
Musuka and co-authors [104]. It is important to mention, that the
commercially available phenothiazines such as for example

  •  trifluoperazine, methdilazine, promazine, promethazine,
    fluphenazin and propiomazine

are beyond patent protection as initially intended. Nevertheless,
these compounds have been patented as adjuvants for the treatment
of MDR cancer (patent expired in 2011 [105]; and, right afterwards,
a new patent has been filed with a priority date of 28th March, 2012,
claiming combination therapy of cancer with a chemotherapeutic
agent and a dopamine receptor antagonist against Cancer stem cells (CSC).

Taking into account that intrinsic MDR is considered as one of the key
properties of CSCs [107], the subject to be covered is indeed related.
According to the MDR, XDR and TDR Mycobacterium tuberculosis,
subjects of this herein paper, the initial step for actually reaching those
in need has been made: a patent has been published in December, 2007,
for the use of TZ and its derivatives for reversing anti-microbial drug
resistance [108]. We must note, however, that, despite the six years
passed since, we were unable to find any related clinical trials, which
would certainly be of outmost importance and urgency in order to
proceed towards an effective therapy of highly resistant mycobacterial

Mechanism Of Action Of Tz: Why It Cures Multi-Drug,
Extensively Drug Resistant And Probably Totally Drug
Resistant Tuberculosis

Over-expressed efflux pumps of Mycobacterium tuberculosis render
the organism multi-drug resistant [13]. Special attention has been
given to those coded by the

  • mmpL7, p55, efpA, mmr, Rv1258c and Rv2459 genes [109].

The activity of these efflux pumps can be suppressed by

  • concentrations of TZ that have no effect on the viability of
    Mycobacterium tuberculosis
  • rendering the organism susceptible to the antibiotic to
    which it was initially resistant
  • as a consequence of the over-expression of its
    efflux pumps [109].

TZ has also been shown to inhibit the activity of the main

  • efflux pumps of bacteria belonging to other species.

TZ has strong inhibitory activity against the genes that code for
essential proteins of M. tuberculosis [122-124].  Consequently, we
may conclude that the in vitro activity of TZ involves

  • the inhibition of the efflux pumps of M. tuberculosis and that
  • the in vitro exposure of this organism to TZ renders the organism
  • susceptible to antibiotics to which it was initially resistant
  • as a consequence of over-expressed efflux pumps [21].

Phenothiazines such as CPZ, TZ, trifluoperazine, etc., also inhibit

  • the binding of calcium to calcium binding proteins such as

calmodulin in eukaryotes [125], and

  • interfere with other proteins involved in
  • the regulation of cellular activity [126].

They inhibit the transport of calcium and potassium systems

  • in eukaryotic cells [127-129] as well as in
  • mycobacteria [89, 130] and
  • E. coli [113].

In fact, in the latter case, calcium was shown essential to

  • the continuous activity of the thioridiazine sensitive
    efflux system [113].

The killing activity of the human macrophage as well as that
of the neutrophil

  • is dependent upon the retention of calcium and potassium
  • within the phagolysosome of the cell [131].

Considering this, several alternative choices are available for
patenting under “new use”, which would allow a “fresh start”
for the compound to be developed. However, the needed
experimental proof that these phenothiazine agents have
activity at the pulmonary macrophage of the alveolar unit
(the site where the causative organism of pulmonary tuberculosis
resides) is still absent.

Targeting the Human Macrophage with Combinations
of Drugs and Inhibitors of Ca2+ and K+ Transport to
Enhance the Killing of Intracellular Multi-Drug Resistant
M. tuberculosis (MDR-TB) – a Novel, Patentable Approach
to Limit the Emergence of XDR-TB

Marta Martins
UCD Centre for Food Safety, School of Agriculture, Food Science and
Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland
& Unit of Mycobacteriology and UPMM; Instituto de Higiene e Medicina
Tropical, Universidade Nova de Lisboa (IHMT/UNL),  Lisbon, Portugal
Recent Patents on Anti-Infective Drug Discovery, 2011, 6, 000-000

The emergence of resistance in Tuberculosis has become a serious
problem for the control of this disease. For that reason, new therapeutic
strategies that can be implemented in the clinical setting are urgently
needed. The design of new compounds active against mycobacteria
must take into account that Tuberculosis is mainly an intracellular
infection of the alveolar macrophage and therefore must maintain
activity within the host cells.

An alternative therapeutic approach will be described in this review,
focusing on the activation of the phagocytic cell and the subsequent
killing of the internalized bacteria. This approach explores the combined
use of antibiotics and phenothiazines, or Ca2+ and K+ flux inhibitors,
in the infected macrophage.

Targeting the infected macrophage and not the internalized bacteria
could overcome the problem of bacterial multi-drug resistance. This
will potentially eliminate the appearance of new multi-drug resistant
tuberculosis (MDR-TB) cases and subsequently prevent the emergence
of extensively-drug resistant tuberculosis (XDR-TB).

Patents resulting from this novel and innovative approach could be
extremely valuable if they can be implemented in the clinical setting.
Other patents will also be discussed such as the treatment of TB
using immunomodulator compounds (for example: betaglycans).

Role of Phenothiazines and Structurally Similar
Compounds of Plant Origin in the Fight against
Infections by Drug Resistant Bacteria

SG. Dastidar 1, JE. Kristiansen 2, J Molnar 3 and L Amaral
Antibiotics 2013; 2: 58-71;

Phenothiazines have their primary effects on the plasma membranes
of prokaryotes and eukaryotes. Among the components of the
prokaryotic plasma membrane affected are

  • efflux pumps,
  • their energy sources
  • and energy providing enzymes, such as ATPase,
  • and genes that regulate and code for the permeability
    aspect of a bacterium.

The response of multidrug and extensively drug resistant
tuberculosis to phenothiazines shows an alternative therapy for the
treatment of these dreaded diseases, which are claiming more and
more lives every year throughout the world.

Many phenothiazines have shown

  • synergistic activity with several antibiotics thereby
  • lowering the doses of antibiotics administered to patients
    suffering from specific bacterial infections.

Trimeprazine is synergistic with trimethoprim. Flupenthixol (Fp)
has been found to be synergistic with penicillin and chlorpromazine
(CPZ); in addition, some antibiotics are also synergistic. Along with
the antibacterial action described in this review,

  • many phenothiazines possess plasmid curing activities, which
  • render the bacterial carrier of the plasmid sensitive to antibiotics.

Thus, simultaneous applications of a phenothiazine like TZ would not
only act as an additional antibacterial agent but also would help

  • to eliminate drug resistant plasmid from
    the infectious bacterial cells.

Part 8.  Cancer Cytotherapy

Synthesis and Structure-Activity Relationships of Novel
Dioxolanes as MDR Modulators in Cancer

A Martins 1,2,†,*, J Csábi 3,†, A Balázs 4, DKitka 1, L Amaral 5,
J Molnár 1, A Simon 6, G Tóth 6 and A Hunyadi 3,
Molecules 2013, 18, 15255-15275;

Ecdysteroids, molting hormones of insects, can exert several mild,
non-hormonal bioactivities in mammals, including humans. In a
previous study, we have found a significant effect of certain derivatives

  • on the ABCB1 transporter mediated multi-drug resistance of a
  • transfected murine leukemia cell line.

In this paper, we present a structure-activity relationship study
focused on

  • the apolar dioxolane derivatives of 20-hydroxyecdysone.

Semi-synthesis and bioactivity of a total of 32 ecdysteroids, including
20 new compounds, is presented, supplemented with their

  • complete 1H- and 13C-NMR signal assignment

As published before [9], the 20,22-diol moiety of 20E is more reactive
than the 2,3-diol, probably due to the free rotation of the 20,22-bond
of 20E that allows the 20,22-dioxolane ring to form with less strain.

This allowed us to selectively obtain the 20,22-mono-dioxolane
derivatives 2–14, or, depending on the amount of reagent and the
reaction time, the 2,3;20,22-bis-homo-dioxolanes 17 and 21–25.

By utilizing the 20,22-monodioxolane ecdysteroids, another aldehyde
or ketone could be coupled to position 2,3, resulting in several bis-hetero-
dioxolane derivatives 26–33. For this, however, gradually decreasing
reactivity with the increase of the size of the reagent was a limiting factor:

  • larger aldehydes or ketones (mainly those containing a
    substituted aromatic ring) could not be coupled at the 2,3-position.
  • The 2,3-monodioxolane derivatives also appeared to be present as
    minor side-products of the reactions, and as a consequence of their
    low amount, only one such compound (compound 15) was isolated and studied.

To selectively obtain this kind of a compound (16) in a more reasonable
yield, another, three-step approach was successfully applied:

  • after protecting the 20,22-diol with phenylboronic acid, the
    2,3-acetonide could be prepared, and
  • removal of the 20,22 protecting group afforded the desired
    2,3-monoacetonide in a one-pot procedure.

In the case of the reactions with aldehydes or asymmetric ketones,
the new C-28 and C-29 central atoms of the dioxolane rings are
stereogenic centers and thus two possible diastereomers can be
formed at both diols. Their configuration was elucidated by two-
dimensional ROESY or selective one-dimensional ROESY experiments,
e.g., in the doubly substituted

  • dioxolane derivative 22 (R1 = R4 = n-Bu, R2 = R3 = H)
  • the unambiguous differentiation of the 1H and 13C signals of
    the two n-butyl groups was achieved in the following way
    (see Figure 2).

Assignment of the H-C(28) atoms (δ = 4.93/105.9 ppm) was supported by

  • the H-2/C-28 and H-3/C-28 HMBC correlations, and
  • that of H-C(29) (δ = 4.91/105.6 ppm) by the H-22/C-29
    cross peak, respectively.

The selective ROESY experiment irradiating at 4.93 ppm showed

  • contacts with the Hα-2 and Hα-3 atoms proving the
    α position of the R2 = H atom.

The ROESY response obtained irradiating H = R3 signal (δ = 4.91)
on H-22 (δ = 3.64 ppm) revealed their

  • cis arrangement and the R configuration around C-29.

The unambiguous assignments of the signals

  • of the two n-butyl groups R1 and R4 were achieved by
  • selective TOCSY experiments (irradiation at
  • δ = 4.93 and 4.91, respectively).

Figure 2

Stereostructure of 22. Red-ROESY proximitries. Blue- 1H. Black-1 001

Stereostructure of 22. Red-ROESY proximitries. Blue- 1H. Black-1 001

Stereostructure of 22. Red arrows indicate the detected ROESY
steric proximities, the blue numbers give the characteristic 1H,
and the black numbers the 13C chemical shifts.


Related Material

Identification of Efflux Pump-mediated Multidrug-resistant
Bacteria by the Ethidium Bromide-agar Cartwheel Method

M Martins, M Viveiros, I Couto, SS. Costa, T Pacheco, S Fanning,
Jean-Marie Pagès, and L Amaral
in vivo 2011; 25: 171-178  

Index for efflux activity of the MDR strains. The capacity to efflux EtBr
of each bacterial strain was ranked relative to the reference strain
according to the following formula:


Index for efflux activity of the MDR strains

Index for efflux activity of the MDR strains

A Simple Method for Assessment of MDR Bacteria for
Over-Expressed Efflux Pumps

M Martinsa,b*, MP. McCuskera,b, M Viveirosa,c, I Coutoc,d,
S Fanninga,b, Jean-Marie Pagès b,e, L Amaral,b,
The Open Microbiol J 2013; 7: 1-5  1874-2858/13 Bentham

Flowchart followed to test bacterial strains using the EtBr-agar
Cartwheel method.

Flowchart followed to test bacterial strains using the EtBr-agar Cartwheel method.

Flowchart followed to test bacterial strains using the EtBr-agar Cartwheel method.

EtBr-agar cartwheel method applied to different bacterial species

EtBr-agar cartwheel method applied to different bacterial species

EtBr-agar cartwheel method applied to different bacterial species

The effect of selected EPIs on the resistance of the induced and
MDR Gram-positive bacteria.

Enterococcus EFC
(4×) (4×) (2×)
                                MCEtBr NOR  (mg/l) MIC NOR (mg/l)
HSEFM-E >2.5 0.125 0.125 0.125 0.125

EPI: Efflux pump inhibitor; w/o: without; TZ: thioridazine; CPZ:
chlorpromazine; PAN: phenyl arginine β-naphthylamide. Values
in bold-type correspond to a decrease of 4-fold or higher on
the MIC values in comparison to those in the absence of inhibitor.
Values in parenthesis indicate the MIC decrease relative to that
of the original culture. The concentration of each EPI used is
defined in the Materials and Methods section.

Macrocyclic diterpenes resensitizing multidrug
resistant phenotypes 

MA. Reis a, A Paterna a, RJ. Ferreira a, H Lage b,
Maria-José U. Ferreira a,⇑
a Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade
de Farmácia, Universidade de Lisboa, Lisboa, Portugal
b Charité Campus Mitte, Institute of Pathology, Berlin, Germany
Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx

Herein, collateral sensitivity effect was exploited as a strategy to
select effective compounds to overcome multidrug resistance in
cancer. Thus, eleven macrocyclic diterpenes, namely jolkinol D (1),
isolated from Euphorbia piscatoria, and its derivatives (2–11) were
evaluated for their activity on three different Human cancer entities:

  • gastric (EPG85-257), pancreatic (EPP85-181) and colon (HT-29)

each with a variant selected for resistance to mitoxantrone

  1. EPG85-257RN;
  2. EPP85-181RN;
  3. HT-29RN and
  • one to daunorubicin (EPG85-257RD; EPP85-181RD; HT-29RD).

Jolkinol D (1) and most of its derivatives (2–11) exhibited significant
collateral sensitivity effect towards the cell lines

  • EPG85-257RN (associated with P-glycoprotein overexpression) and
  • HT-29RD (altered topoisomerase II expression).

The benzoyl derivative, jolkinoate L (8) demonstrated ability to

  • target different cellular contexts with
  • concomitant high antiproliferative activity.

These compounds were previously assessed as
P-glycoprotein modulators,

  • at non-cytotoxic doses, on MDR1-mouse lymphoma cells.

A regression analysis between

  1. the antiproliferative activity presented herein and
  2. the previously assessed P-glycoprotein modulatory effect

showed a strong relation between the compounds that presented

  • both high P-glycoprotein modulation and cytotoxicity.

Molecular Docking Characterizes Substrate-Binding Sites
and Efflux Modulation Mechanisms within P

Ferreira,† Maria-José U. Ferreira,† and DJVA dos Santos*,†,‡
†Research Institute for Medicines and Pharmaceutical Sciences
(iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal
‡REQUIMTE, Department of Chemistry & Biochemistry, Faculty of
Sciences, University of Porto, Porto, Portugal
J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

P-Glycoprotein (Pgp) is one of the best characterized ABC
, often involved

  • in the multidrug-resistance phenotype
  • overexpressed by several cancer cell lines.

Experimental studies contributed to important knowledge concerning
substrate polyspecificity, efflux mechanism, and drug binding sites.
This information is, however, scattered through different perspectives,
not existing a unifying model for the knowledge available for this transporter.
Using a previously refined structure of murine Pgp,

  • three putative drug-binding sites were hereby characterized
  • by means of molecular docking.

The modulator site (M-site) is characterized by

  • cross interactions between both Pgp halves

herein defined for the first time, having an important role in

  • impairing conformational changes leading to substrate efflux.

Two other binding sites, located next to the inner leaflet of the lipid bilayer,

  • were identified as the substrate binding H and R sites
  • by matching docking and experimental results.

A new classification model

  • with the ability to discriminate substrates from modulators

is also proposed, integrating a vast number of theoretical and experimental data.

conformational changes leading to substrate efflux

conformational changes leading to substrate efflux

conformational changes leading to substrate efflux



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Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief

Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief

Reviewer and Curator: Larry H. Bernstein, MD, FCAP

Pentose Shunt, Electron Transfer, Galactose, and other Lipids in brief

This is a continuation of the series of articles that spans the horizon of the genetic
code and the progression in complexity from genomics to proteomics, which must
be completed before proceeding to metabolomics and multi-omics.  At this point
we have covered genomics, transcriptomics, signaling, and carbohydrate metabolism
with considerable detail.In carbohydrates. There are two topics that need some attention –
(1) pentose phosphate shunt;
(2) H+ transfer
(3) galactose.
(4) more lipids
Then we are to move on to proteins and proteomics.

Summary of this series:

The outline of what I am presenting in series is as follows:

  1. Signaling and Signaling Pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism

Selected References to Signaling and Metabolic Pathways published in this Open Access Online Scientific Journal, include the following:

  1. Lipid metabolism

4.1  Studies of respiration lead to Acetyl CoA

4.2 The multi-step transfer of phosphate bond and hydrogen exchange energy

5.Pentose shunt, electron transfers, galactose, and other lipids in brief

6. Protein synthesis and degradation

7.  Subcellular structure

8. Impairments in pathological states: endocrine disorders; stress
hypermetabolism; cancer.

Section I. Pentose Shunt

Bernard L. Horecker’s Contributions to Elucidating the Pentose Phosphate Pathway

Nicole Kresge,     Robert D. Simoni and     Robert L. Hill

The Enzymatic Conversion of 6-Phosphogluconate to Ribulose-5-Phosphate
and Ribose-5-Phosphate (Horecker, B. L., Smyrniotis, P. Z., and Seegmiller,
J. E.      J. Biol. Chem. 1951; 193: 383–396

Bernard Horecker

Bernard Leonard Horecker (1914) began his training in enzymology in 1936 as a
graduate student at the University of Chicago in the laboratory of T. R. Hogness.
His initial project involved studying succinic dehydrogenase from beef heart using
the Warburg manometric apparatus. However, when Erwin Hass arrived from Otto
Warburg’s laboratory he asked Horecker to join him in the search for an enzyme
that would catalyze the reduction of cytochrome c by reduced NADP. This marked
the beginning of Horecker’s lifelong involvement with the pentose phosphate pathway.

During World War II, Horecker left Chicago and got a job at the National Institutes of
Health (NIH) in Frederick S. Brackett’s laboratory in the Division of Industrial Hygiene.
As part of the wartime effort, Horecker was assigned the task of developing a method
to determine the carbon monoxide hemoglobin content of the blood of Navy pilots
returning from combat missions. When the war ended, Horecker returned to research
in enzymology and began studying the reduction of cytochrome c by the succinic
dehydrogenase system.

Shortly after he began these investigation changes, Horecker was approached by
future Nobel laureate Arthur Kornberg, who was convinced that enzymes were the
key to understanding intracellular biochemical processes
. Kornberg suggested
they collaborate, and the two began to study the effect of cyanide on the succinic
dehydrogenase system. Cyanide had previously been found to inhibit enzymes
containing a heme group, with the exception of cytochrome c. However, Horecker
and Kornberg found that

  • cyanide did in fact react with cytochrome c and concluded that
  • previous groups had failed to perceive this interaction because
    • the shift in the absorption maximum was too small to be detected by
      visual examination.

Two years later, Kornberg invited Horecker and Leon Heppel to join him in setting up
a new Section on Enzymes in the Laboratory of Physiology at the NIH. Their Section on Enzymes eventually became part of the new Experimental Biology and Medicine
Institute and was later renamed the National Institute of Arthritis and Metabolic

Horecker and Kornberg continued to collaborate, this time on

  • the isolation of DPN and TPN.

By 1948 they had amassed a huge supply of the coenzymes and were able to
present Otto Warburg, the discoverer of TPN, with a gift of 25 mg of the enzyme
when he came to visit. Horecker also collaborated with Heppel on 

  • the isolation of cytochrome c reductase from yeast and 
  • eventually accomplished the first isolation of the flavoprotein from
    mammalian liver.

Along with his lab technician Pauline Smyrniotis, Horecker began to study

  • the enzymes involved in the oxidation of 6-phosphogluconate and the
    metabolic intermediates formed in the pentose phosphate pathway.

Joined by Horecker’s first postdoctoral student, J. E. Seegmiller, they worked
out a new method for the preparation of glucose 6-phosphate and 6-phosphogluconate, 
both of which were not yet commercially available.
As reported in the Journal of Biological Chemistry (JBC) Classic reprinted here, they

  • purified 6-phosphogluconate dehydrogenase from brewer’s yeast (1), and 
  • by coupling the reduction of TPN to its reoxidation by pyruvate in
    the presence of lactic dehydrogenase
  • they were able to show that the first product of 6-phosphogluconate oxidation,
  • in addition to carbon dioxide, was ribulose 5-phosphte.
  • This pentose ester was then converted to ribose 5-phosphate by a
    pentose-phosphate isomerase.

They were able to separate ribulose 5-phosphate from ribose 5- phosphate and demonstrate their interconversion using a recently developed nucleotide separation
technique called ion-exchange chromatography. Horecker and Seegmiller later
showed that 6-phosphogluconate metabolism by enzymes from mammalian
tissues also produced the same products

Bernard Horecker

Bernard Horecker

Over the next several years, Horecker played a key role in elucidating the

  • remaining steps of the pentose phosphate pathway.

His total contributions included the discovery of three new sugar phosphate esters,
ribulose 5-phosphate, sedoheptulose 7-phosphate, and erythrose 4-phosphate, and
three new enzymes, transketolase, transaldolase, and pentose-phosphate 3-epimerase.
The outline of the complete pentose phosphate cycle was published in 1955
(2). Horecker’s personal account of his work on the pentose phosphate pathway can
be found in his JBC Reflection (3).1

Horecker’s contributions to science were recognized with many awards and honors
including the Washington Academy of Sciences Award for Scientific Achievement in
Biological Sciences (1954) and his election to the National Academy of Sciences in
1961. Horecker also served as president of the American Society of Biological
Chemists (now the American Society for Biochemistry and Molecular Biology) in 1968.


  • 1 All biographical information on Bernard L. Horecker was taken from Ref. 3.
  • The American Society for Biochemistry and Molecular Biology, Inc.


  1. ↵Horecker, B. L., and Smyrniotis, P. Z. (1951) Phosphogluconic acid dehydrogenase
    from yeast. J. Biol. Chem. 193, 371–381FREE Full Text
  2. Gunsalus, I. C., Horecker, B. L., and Wood, W. A. (1955) Pathways of carbohydrate
    metabolism in microorganisms. Bacteriol. Rev. 19, 79–128  FREE Full Text
  3. Horecker, B. L. (2002) The pentose phosphate pathway. J. Biol. Chem. 277, 47965–
    47971 FREE Full Text

The Pentose Phosphate Pathway (also called Phosphogluconate Pathway, or Hexose
Monophosphate Shunt) is depicted with structures of intermediates in Fig. 23-25
p. 863 of Biochemistry, by Voet & Voet, 3rd Edition. The linear portion of the pathway
carries out oxidation and decarboxylation of glucose-6-phosphate, producing the
5-C sugar ribulose-5-phosphate.

Glucose-6-phosphate Dehydrogenase catalyzes oxidation of the aldehyde
(hemiacetal), at C1 of glucose-6-phosphate, to a carboxylic acid in ester linkage
(lactone). NADPserves as electron acceptor.

6-Phosphogluconolactonase catalyzes hydrolysis of the ester linkage (lactone)
resulting in ring opening. The product is 6-phosphogluconate. Although ring opening
occurs in the absence of a catalyst, 6-Phosphogluconolactonase speeds up the
reaction, decreasing the lifetime of the highly reactive, and thus potentially
toxic, 6-phosphogluconolactone.

Phosphogluconate Dehydrogenase catalyzes oxidative decarboxylation of
6-phosphogluconate, to yield the 5-C ketose ribulose-5-phosphate. The
hydroxyl at C(C2 of the product) is oxidized to a ketone. This promotes loss
of the carboxyl at C1 as CO2.  NADP+ again serves as oxidant (electron acceptor).

pglucose hd

pglucose hd

Reduction of NADP+ (as with NAD+) involves transfer of 2e- plus 1H+ to the
nicotinamide moiety.


NADPH, a product of the Pentose Phosphate Pathway, functions as a reductant in
various synthetic (anabolic) pathways, including fatty acid synthesis.

NAD+ serves as electron acceptor in catabolic pathways in which metabolites are
oxidized. The resultant NADH is reoxidized by the respiratory chain, producing ATP.


Glucose-6-phosphate Dehydrogenase is the committed step of the Pentose
Phosphate Pathway. This enzyme is regulated by availability of the substrate NADP+.
As NADPH is utilized in reductive synthetic pathways, the increasing concentration of
NADP+ stimulates the Pentose Phosphate Pathway, to replenish NADPH.

The remainder of the Pentose Phosphate Pathway accomplishes conversion of the
5-C ribulose-5-phosphate to the 5-C product ribose-5-phosphate, or to the 3-C
glyceraldehyde -3-phosphate and the 6-C fructose-6-phosphate (reactions 4 to 8
p. 863).

Transketolase utilizes as prosthetic group thiamine pyrophosphate (TPP), a
derivative of vitamin B1.



Thiamine pyrophosphate binds at the active sites of enzymes in a “V” conformation.The amino group of the aminopyrimidine moiety is close to the dissociable proton,
and serves as the proton acceptor. This proton transfer is promoted by a glutamate
residue adjacent to the pyrimidine ring.

The positively charged N in the thiazole ring acts as an electron sink, promoting
C-C bond cleavage. The 3-C aldose glyceraldehyde-3-phosphate is released.
2-C fragment remains on TPP.

FASEB J. 1996 Mar;10(4):461-70.


The importance of this pathway can easily be underestimated.  The main source for
energy in respiration was considered to be tied to the

  • high energy phosphate bond in phosphorylation and utilizes NADPH, converting it to NADP+.

glycolysis n skeletal muscle in short term, dependent on muscle glycogen conversion
to glucose, and there is a buildup of lactic acid – used as fuel by the heart.  This
pathway accounts for roughly 5% of metabolic needs, varying between tissues,
depending on there priority for synthetic functions, such as endocrine or nucleic
acid production.

The mature erythrocyte and the ocular lens both are enucleate.  85% of their
metabolic energy needs are by anaerobic glycolysis.  Consider the erythrocyte
somewhat different than the lens because it has iron-based hemoglobin, which
exchanges O2 and CO2 in the pulmonary alveoli, and in that role, is a rapid
regulator of H+ and pH in the circulation (carbonic anhydrase reaction), and also to
a lesser extent in the kidney cortex, where H+ is removed  from the circulation to
the urine, making the blood less acidic, except when there is a reciprocal loss of K+.
This is how we need a nomogram to determine respiratory vs renal acidosis or
alkalosis.  In the case of chronic renal disease, there is substantial loss of
functioning nephrons, loss of countercurrent multiplier, and a reduced capacity to
remove H+.  So there is both a metabolic acidosis and a hyperkalemia, with increased
serum creatinine, but the creatinine is only from muscle mass – not accurately
reflecting total body mass, which includes visceral organs.  The only accurate
measure of lean body mass would be in the linear relationship between circulating
hepatic produced transthyretin (TTR).

The pentose phosphate shunt is essential for

  • the generation of nucleic acids, in regeneration of red cells and lens – requiring NADPH.

Insofar as the red blood cell is engaged in O2 exchange, the lactic dehydrogenase
isoenzyme composition is the same as the heart. What about the lens of and cornea the eye, and platelets?  The explanation does appear to be more complex than
has been proposed and is not discussed here.

Section II. Mitochondrial NADH – NADP+ Transhydrogenase Reaction

There is also another consideration for the balance of di- and tri- phospopyridine
nucleotides in their oxidized and reduced forms.  I have brought this into the
discussion because of the centrality of hydride tranfer to mitochondrial oxidative
phosphorylation and the energetics – for catabolism and synthesis.

The role of transhydrogenase in the energy-linked reduction of TPN 

Fritz HommesRonald W. Estabrook∗∗

The Wenner-Gren Institute, University of Stockholm
Stockholm, Sweden
Biochemical and Biophysical Research Communications 11, (1), 2 Apr 1963, Pp 1–6

In 1959, Klingenberg and Slenczka (1) made the important observation that incubation of isolated

  • liver mitochondria with DPN-specific substrates or succinate in the absence of phosphate
    acceptor resulted in a rapid and almost complete reduction of the intramitochondrial TPN.

These and related findings led Klingenberg and co-workers (1-3) to postulate

  • the occurrence of an ATP-controlled transhydrogenase reaction catalyzing the reduction of
    mitochondrial TPN by DPNH. A similar conclusion was reached by Estabrook and Nissley (4).

The present paper describes the demonstration and some properties of an

  • energy-dependent reduction of TPN by DPNH, catalyzed by submitochondrial particles.

Preliminary reports of some of these results have already appeared (5, 6 ) , and a
complete account is being published elsewhere (7).We have studied the energy- dependent reduction of TPN by PNH with submitochondrial particles from both
rat liver and beef heart. Rat liver particles were prepared essentially according to
the method of Kielley and Bronk (8), and beef heart particles by the method of
Low and Vallin (9).


(From the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore,
Maryland)  J. Biol. Chem. 1952, 195:107-119.

NO Kaplan

NO Kaplan

Sidney Colowick

Sidney Colowick

Elizabeth Neufeld

Elizabeth Neufeld

Kaplan studied carbohydrate metabolism in the liver under David M. Greenberg at the
University of California, Berkeley medical school. He earned his Ph.D. in 1943. From
1942 to 1944, Kaplan participated in the Manhattan Project. From 1945 to 1949,
Kaplan worked with Fritz Lipmann at Massachusetts General Hospital to study
coenzyme A. He worked at the McCollum-Pratt Institute of Johns Hopkins University
from 1950 to 957. In 1957, he was recruited to head a new graduate program in
biochemistry at Brandeis University. In 1968, Kaplan moved to the University of
California, San Diego
, where he studied the role of lactate dehydrogenase in cancer. He also founded a colony of nude mice, a strain of laboratory mice useful in the study
of cancer and other diseases. [1] He was a member of the National Academy of
Sciences.One of Kaplan’s students at the University of California was genomic
researcher Craig Venter.[2]3]  He was, with Sidney Colowick, a founding editor of the scientific book series Methods
in Enzymology

Colowick became Carl Cori’s first graduate student and earned his Ph.D. at
Washington University St. Louis in 1942, continuing to work with the Coris (Nobel
Prize jointly) for 10 years. At the age of 21, he published his first paper on the
classical studies of glucose 1-phosphate (2), and a year later he was the sole author on a paper on the synthesis of mannose 1-phosphate and galactose 1-phosphate (3). Both papers were published in the JBC. During his time in the Cori lab,

Colowick was involved in many projects. Along with Herman Kalckar he discovered
myokinase (distinguished from adenylate kinase from liver), which is now known as
adenyl kinase. This discovery proved to be important in understanding transphos-phorylation reactions in yeast and animal cells. Colowick’s interest then turned to
the conversion of glucose to polysaccharides, and he and Earl Sutherland (who
will be featured in an upcoming JBC Classic) published an important paper on the
formation of glycogen from glucose using purified enzymes (4). In 1951, Colowick
and Nathan Kaplan were approached by Kurt Jacoby of Academic Press to do a
series comparable to Methodem der Ferment Forschung. Colowick and Kaplan
planned and edited the first 6 volumes of Methods in Enzymology, launching in 1955
what became a series of well known and useful handbooks. He continued as
Editor of the series until his death in 1985.

The Structure of NADH: the Work of Sidney P. Colowick

Nicole KresgeRobert D. Simoni and Robert L. Hill

On the Structure of Reduced Diphosphopyridine Nucleotide

(Pullman, M. E., San Pietro, A., and Colowick, S. P. (1954)

J. Biol. Chem. 206, 129–141)

Elizabeth Neufeld
·  Born: September 27, 1928 (age 85), Paris, France
·  EducationQueens College, City University of New YorkUniversity of California,

In Paper I (l), indirect evidence was presented for the following transhydrogenase
reaction, catalyzed by an enzyme present in extracts of Pseudomonas


The evidence was obtained by coupling TPN-specific dehydrogenases with the
transhydrogenase and observing the reduction of large amounts of diphosphopyridine nucleotide (DPN) in the presence of catalytic amounts of triphosphopyridine
nucleotide (TPN).

In this paper, data will be reported showing the direct

  • interaction between TPNHz and DPN, in thepresence of transhydrogenase alone,
  • to yield products having the propertiesof TPN and DPNHZ.

Information will be given indicating that the reaction involves

  • a transfer of electrons (or hydrogen) rather than a phosphate 

Experiments dealing with the kinetics and reversibility of the reaction, and with the
nature of the products, suggest that the reaction is a complex one, not fully described
by the above formulation.

Materials and Methods [edited]

The TPN and DPN used in these studies were preparations of approximately 75
percent purity and were prepared from sheep liver by the chromatographic procedure
of Kornberg and Horecker (unpublished). Reduced DPN was prepared enzymatically with alcohol dehydrogenase as described elsewhere (2). Reduced TPN was prepared by treating TPN with hydrosulfite. This treated mixture contained 2 pM of TPNHz per ml.
The preparations of desamino DPN and reduced desamino DPN have been
described previously (2, 3). Phosphogluconate was a barium salt which was kindly
supplied by Dr. B. F. Horecker. Cytochrome c was obtained from the Sigma Chemical Company.

Transhydrogenase preparations with an activity of 250 to 7000 units per mg. were
used in these studies. The DPNase was a purified enzyme, which was obtained
from zinc-deficient Neurospora and had an activity of 5500 units per mg. (4). The
alcohol dehydrogenase was a crystalline preparation isolated from yeast according to the procedure of Racker (5).

Phosphogluconate dehydrogenase from yeast and a 10 per cent pure preparation of the TPN-specific cytochrome c reductase from liver (6) were gifts of Dr. B. F.

DPN was assayed with alcohol and crystalline yeast alcohol dehydrogenase. TPN was determined By the specific phosphogluconic acid dehydrogenase from yeast and also by the specific isocitric dehydrogenase from pig heart. Reduced DPN was
determined by the use of acetaldehyde and the yeast alcohol dehydrogenase.
All of the above assays were based on the measurement of optical density changes
at 340 rnp. TPNHz was determined with the TPN-specific cytochrome c reductase system. The assay of the reaction followed increase in optical density at 550 rnp  as a measure of the reduction of the cytochrome c after cytochrome c
reductase was added to initiate the reaction. The changes at 550 rnp are plotted for different concentrations of TPNHz in Fig. 3, a. The method is an extremely sensitive and accurate assay for reduced TPN.

[No Figures or Table shown]

Formation of DPNHz from TPNHz and DPN-Fig. 1, a illustrates the direct reaction between TPNHz and DPN to form DPNHZ. The reaction was carried out by incubating TPNHz with DPN in the presence of the
transhydrogenase, yeast alcohol dehydrogenase, and acetaldehyde. Since the yeast dehydrogenase is specific for DPN,

  • a decrease in absorption at340 rnp can only be due to the formation of reduced DPN. It can
    be seen from the curves in Fig. 1, a that a decrease in optical density occurs only in the
    presence of the complete system.

The Pseudomonas enzyme is essential for the formation of DPNH2. It is noteworthy
that, under the conditions of reaction in Fig. 1, a,

  • approximately 40 per cent of theTPNH, reacted with the DPN.

Fig. 1, a also indicates that magnesium is not required for transhydrogenase activity.  The reaction between TPNHz and DPN takes place in the absence of alcohol
dehydrogenase and acetaldehyde
. This can be demonstrated by incubating the
two pyridine nucleotides with the transhydrogenase for 4 8 12 16 20 24 28 32 36

FIG. 1. Evidence for enzymatic reaction of TPNHt with DPN.

  • Rate offormation of DPNH2.

(b) DPN disappearance and TPN formation.

(c) Identification of desamino DPNHz as product of reaction of TPNHz with desamino DPN.  (assaying for reduced DPN by the yeast alcohol dehydrogenase technique.

Table I (Experiment 1) summarizes the results of such experiments in which TPNHz was added with varying amounts of DPN.

  • In the absence of DPN, no DPNHz was formed. This eliminates the possibility that TPNH 2 is
    converted to DPNHz
  • by removal ofthe monoester phosphate grouping.

The data also show that the extent of the reaction is

  • dependent on the concentration of DPN.

Even with a large excess of DPN, only approximately 40 per cent of the TPNHzreacts to form reduced DPN. It is of importance to emphasize that in the above
experiments, which were carried out in phosphate buffer, the extent of  the reaction

  • is the same in the presence or absence of acetaldehyde andalcohol dehydrogenase.

With an excess of DPN and different  levels of TPNHZ,

  • the amount of reduced DPN which is formed is
  • dependent on the concentration of TPNHz(Table I, Experiment 2).
  • In all cases, the amount of DPNHz formed is approximately
    40 per cent of the added reduced TPN.

Formation of TPN-The reaction between TPNHz and DPN should yield TPN as well as DPNHz.
The formation of TPN is demonstrated in Table 1. in Fig. 1, b. In this experiment,
TPNHz was allowed to react with DPN in the presence of the transhydrogenase
(PS.), and then alcohol and alcohol dehydrogenase were added . This
would result in reduction of the residual DPN, and the sample incubated with the
transhydrogenase contained less DPN. After the completion of the alcohol
dehydrogenase reaction, phosphogluconate and phosphogluconic dehydrogenase (PGAD) were added to reduce the TPN. The addition of this TPN-specific
dehydrogenase results in an

  • increase inoptical density in the enzymatically treated sample.
  • This change represents the amount of TPN formed.

It is of interest to point out that, after addition of both dehydrogenases,

  • the total optical density change is the same in both

Therefore it is evident that

  • for every mole of DPN disappearing  a mole of TPN appears.

Balance of All Components of Reaction

Table II (Experiment 1) shows that,

  • if measurements for all components of the reaction are made, one can demonstrate
    that there is
  • a mole for mole disappearance of TPNH, and DPN, and
  • a stoichiometric appearance of TPN and DPNH2.
  1. The oxidized forms of the nucleotides were assayed as described
  2. the reduced form of TPN was determined by the TPNHz-specific cytochrome c reductase,
  3. the DPNHz by means of yeast alcohol dehydrogenase plus

This stoichiometric balance is true, however,

  • only when the analyses for the oxidized forms are determined directly on the reaction

When analyses are made after acidification of the incubated reaction mixture,

  • the values found forDPN and TPN are much lower than those obtained by direct analysis.

This discrepancy in the balance when analyses for the oxidized nucleotides are
carried out in acid is indicated in Table II (Experiment 2). The results, when
compared with the findings in Experiment 1, are quite striking.

Reaction of TPNHz with Desamino DPN

Desamino DPN

  • reacts with the transhydrogenase system at the same rate as does DPN (2).

This was of value in establishing the fact that

  • the transhydrogenase catalyzesa transfer of hydrogen rather than a phosphate transfer reaction.

The reaction between desamino DPN and TPNHz can be written in two ways.

TPN f desamino DPNHz

TPNH, + desamino DPN

DPNH2 + desamino TPN

If the reaction involved an electron transfer,

  • desamino DPNHz would be
  • Phosphate transfer would result in the production of reduced

Desamino DPNHz can be distinguished from DPNHz by its

  • slowerrate of reaction with yeast alcohol dehydrogenase (2, 3).

Fig. 1, c illustrates that, when desamino DPN reacts with TPNH2, 

  • the product of the reaction is desamino DPNHZ.

This is indicated by the slow rate of oxidation of the product by yeast alcohol
dehydrogenase and acetaldehyde.

From the above evidence phosphate transfer 

  • has been ruled out as a possible mechanism for the transhydrogenase reaction.

Inhibition by TPN

As mentioned in Paper I and as will be discussed later in this paper,

  • the transhydrogenase reaction does not appear to be readily reversible.

This is surprising, particularly since only approximately 

  • 40 per cent of the TPNHz undergoes reaction with DPN
    under the conditions described above. It was therefore thought that
  • the TPN formed might inhibit further transfer of electrons from TPNH2.

Table III summarizes data showing the

  • strong inhibitory effect of TPN on thereaction between TPNHz and DPN.

It is evident from the data that

  • TPN concentration is a factor in determining the extent of the reaction.

Effect of Removal of TPN on Extent of Reaction

A purified DPNase from Neurospora has been found

  • to cleave the nicotinamide riboside linkagesof the oxidized forms of both TPN and DPN
  • without acting on thereduced forms of both nucleotides (4).

It has been found, however, that

  • the DPNase hydrolyzes desamino DPN at a very slow rate (3).

In the reaction between TPNHz and desamino DPN, TPN and desamino DPNH:,

  • TPNis the only component of this reaction attacked by the Neurospora enzyme
    at an appreciable rate

It was  thought that addition of the DPNase to the TPNHZ-desamino DPN trans-
hydrogenase reaction mixture

  • would split the TPN formed andpermit the reaction to go to completion.

This, indeed, proved to be the case, as indicated in Table IV, where addition of
the DPNase with desamino DPN results in almost

  • a stoichiometric formation of desamino DPNHz
  • and a complete disappearance of TPNH2.

Extent of Reaction in Buffers Other Than Phosphate

All the reactions described above were carried out in phosphate buffer of pH 7.5.
If the transhydrogenase reaction between TPNHz and DPN is run at the same pH
in tris(hydroxymethyl)aminomethane buffer (TRIS buffer)

  • with acetaldehydeand alcohol dehydrogenase present,
  • the reaction proceeds muchfurther toward completion 
  • than is the case under the same conditions ina phosphate medium (Fig. 2, a).

The importance of phosphate concentration in governing the extent of the reaction
is illustrated in Fig. 2, b.

In the presence of TRIS the transfer reaction

  • seems to go further toward completion in the presence of acetaldehyde
    alcohol dehydrogenase
  • than when these two components are absent.

This is not true of the reaction in phosphate,

  • in which the extent is independent of the alcoholdehydrogenase system.

Removal of one of the products of the reaction (DPNHp) in TRIS thus

  • appears to permit the reaction to approach completion,whereas
  • in phosphate this removal is without effect on the finalcourse of the reaction.

The extent of the reaction in TRIS in the absence of alcohol dehydrogenase
and acetaldehyde

  • somewhat greater than when the reaction is run in phosphate.

TPN also inhibits the reaction of TPNHz with DPN in TRIS medium, but the inhibition

  • is not as marked as when the reaction is carried out in phosphate buffer.

Reversibility of Transhydrogenase Reaction;

Reaction between DPNHz and TPN

In Paper I, it was mentioned that no reversal of the reaction could be achieved in a system containing alcohol, alcohol dehydrogenase, TPN, and catalytic amounts of

When DPNH, and TPN are incubated with the purified transhydrogenase, there is

  • no evidence for reversibility.

This is indicated in Table V which shows that

  • there is no disappearance of DPNHz in such a system.

It was thought that removal of the TPNHz, which might be formed in the reaction,
could promote the reversal of the reaction. Hence,

  • by using the TPNHe-specific cytochrome c reductase, one could
  1. not only accomplishthe removal of any reduced TPN,
  2. but also follow the course of the reaction.

A system containing DPNH2, TPN, the transhydrogenase, the cytochrome c
reductase, and cytochrome c, however, gives

  • no reduction of the cytochrome

This is true for either TRIS or phosphate buffers.2

Some positive evidence for the reversibility has been obtained by using a system

  • DPNH2, TPNH2, cytochrome c, and the cytochrome creductase in TRIS buffer.

In this case, there is, of course, reduction of cytochrome c by TPNHZ, but,

  • when the transhydrogenase is present.,there is
  • additional reduction over and above that due to the added TPNH2.

This additional reduction suggests that some reversibility of the reaction occurred
under these conditions. Fig. 3, b shows

  • the necessity of DPNHzfor this additional reduction.

Interaction of DPNHz with Desamino DPN-

If desamino DPN and DPNHz are incubated with the purified Pseudomonas enzyme,
there appears

  • to be a transfer of electrons to form desamino DPNHz.

This is illustrated in Fig. 4, a, which shows the

  • decreased rate of oxidation by thealcohol dehydrogenase system
  • after incubation with the transhydrogenase.
  • Incubation of desamino DPNHz with DPN results in the formation of DPNH2,
  • which is detected by the faster rate of oxidation by the alcohol dehydrogenase system
  • after reaction of the pyridine nucleotides with thetranshydrogenase (Fig. 4, b).

It is evident from the above experiments that

the transhydrogenase catalyzes an exchange of hydrogens between

  • the adenylic and inosinic pyridine nucleotides.

However, it is difficult to obtain any quantitative information on the rate or extent of
the reaction by the method used, because

  • desamino DPNHz also reacts with the alcohol dehydrogenase system,
  • although at a much slower rate than does DPNH2.


The results of the balance experiments seem to offer convincing evidence that
the transhydrogenase catalyzes the following reaction.


Since desamino DPNHz is formed from TPNHz and desamino DPN,

  • thereaction appears to involve an electron (or hydrogen) transfer
  • rather thana transfer of the monoester phosphate grouping of TPN.

A number of the findings reported in this paper are not readily understandable in
terms of the above simple formulation of the reaction. It is difficult to understand
the greater extent of the reaction in TRIS than in phosphate when acetaldehyde
and alcohol dehydrogenase are present.

One possibility is that an intermediate may be involved which is more easily converted
to reduced DPN in the TRIS medium. The existence of such an intermediate is also
suggested by the discrepancies noted in balance experiments, in which

  • analyses of the oxidized nucleotides after acidification showed
  • much lower values than those found by direct analysis.

These findings suggest that the reaction may involve

  • a 1 electron ratherthan a 2 electron transfer with
  • the formation of acid-labile free radicals as intermediates.

The transfer of hydrogens from DPNHz to desamino DPN

  • to yield desamino DPNHz and DPN and the reversal of this transfer
  • indicate the unique role of the transhydrogenase
  • in promoting electron exchange between the pyridine nucleotides.

In this connection, it is of interest that alcohol dehydrogenase and lactic
dehydrogenase cannot duplicate this exchange  between the DPN and
the desamino systems.3  If one assumes that desamino DPN behaves
like DPN,

  • one might predict that the transhydrogenase would catalyze an
    exchange of electrons (or hydrogen) 3.

Since alcohol dehydrogenase alone

  • does not catalyze an exchange of electrons between the adenylic
    and inosinic pyridine nucleotides, this rules out the possibility
  • that the dehydrogenase is converted to a reduced intermediate
  • during electron between DPNHz and added DPN.

It is hoped to investigate this possibility with isotopically labeled DPN.
Experiments to test the interaction between TPN and desamino TPN are
also now in progress.

It seems likely that the transhydrogenase will prove capable of

  • catalyzingan exchange between TPN and TPNH2, as well as between DPN and

The observed inhibition by TPN of the reaction between TPNHz and DPN may

  • be due to a competition between DPN and TPNfor the TPNH2.


  1. Direct evidence for the following transhydrogenase reaction. catalyzedby an
    enzyme from Pseudomonas fluorescens, is presented.


Balance experiments have shown that for every mole of TPNHz disappearing
1 mole of TPN appears and that for each mole of DPNHz generated 1 mole of
DPN disappears. The oxidized nucleotides found at the end of the reaction,
however, show anomalous lability toward acid.

  1. The transhydrogenase also promotes the following reaction.

TPNHz + desamino DPN -+ TPN + desamino DPNH,

This rules out the possibility that the transhydrogenase reaction involves a
phosphate transfer and indicates that the

  • enzyme catalyzes a shift of electrons (or hydrogen atoms).

The reaction of TPNHz with DPN in 0.1 M phosphate buffer is strongly
inhibited by TPN; thus

  • it proceeds only to the extent of about40 per cent or less, even
  • when DPNHz is removed continuously by meansof acetaldehyde
    and alcohol dehydrogenase.
  • In other buffers, in whichTPN is less inhibitory, the reaction proceeds
    much further toward completion under these conditions.
  • The reaction in phosphate buffer proceedsto completion when TPN
    is removed as it is formed.
  1. DPNHz does not react with TPN to form TPNHz and DPN in the presence
    of transhydrogenase. Some evidence, however, has been obtained for
    the reversibility by using the following system:
  • DPNHZ, TPNHZ, cytochromec, the TPNHz-specific cytochrome c reductase,
    and the transhydrogenase.
  1. Evidence is cited for the following reversible reaction, which is catalyzed
    by the transhydrogenase.

DPNHz + desamino DPN fi DPN + desamino DPNHz

  1. The results are discussed with respect to the possibility that the
    transhydrogenase reaction may
  • involve a 1 electron transfer with theformation of free radicals as intermediates.



  1. Coiowick, S. P., Kaplan, N. O., Neufeld, E. F., and Ciotti, M. M., J. Biol. Chem.,196, 95 (1952).
  2. Pullman, 111. E., Colowick, S. P., and Kaplan, N. O., J. Biol. Chem., 194, 593(1952).
  3. Kaplan, N. O., Colowick, S. P., and Ciotti, M. M., J. Biol. Chem., 194, 579 (1952).
  4. Kaplan, N. O., Colowick, S. P., and Nason, A., J. Biol. Chem., 191, 473 (1951).
  5. Racker, E., J. Biol. Chem., 184, 313 (1950).
  6. Horecker, B. F., J. Biol. Chem., 183, 593 (1950).

Section !II. 


The Leloir pathway: a mechanistic imperative for three enzymes to change
the stereochemical configuration of a single carbon in galactose.

Frey PA.
FASEB J. 1996 Mar;10(4):461-70.

The biological interconversion of galactose and glucose takes place only by way of
the Leloir pathway and requires the three enzymes galactokinase, galactose-1-P
uridylyltransferase, and UDP-galactose 4-epimerase.
The only biological importance of these enzymes appears to be to

  • provide for the interconversion of galactosyl and glucosyl groups.

Galactose mutarotase also participates by producing the galactokinase substrate
alpha-D-galactose from its beta-anomer. The galacto/gluco configurational change takes place at the level of the nucleotide sugar by an oxidation/reduction
mechanism in the active site of the epimerase NAD+ complex. The nucleotide portion
of UDP-galactose and UDP-glucose participates in the epimerization process in two ways:

1) by serving as a binding anchor that allows epimerization to take place at glycosyl-C-4 through weak binding of the sugar, and

2) by inducing a conformational change in the epimerase that destabilizes NAD+ and
increases its reactivity toward substrates.

Reversible hydride transfer is thereby facilitated between NAD+ and carbon-4
of the weakly bound sugars.

The structure of the enzyme reveals many details of the binding of NAD+ and
inhibitors at the active site

The essential roles of the kinase and transferase are to attach the UDP group
to galactose, allowing for its participation in catalysis by the epimerase. The
transferase is a Zn/Fe metalloprotein
, in which the metal ions stabilize the
structure rather than participating in catalysis. The structure is interesting
in that

  • it consists of single beta-sheet with 13 antiparallel strands and 1 parallel strand
    connected by 6 helices.

The mechanism of UMP attachment at the active site of the transferase is a double
, with the participation of a covalent UMP-His 166-enzyme intermediate
in the Escherichia coli enzyme. The evolution of this mechanism appears to have
been guided by the principle of economy in the evolution of binding sites.

PMID: 8647345 Free full text

Section IV.

More on Lipids – Role of lipids – classification

  • Energy
  • Energy Storage
  • Hormones
  • Vitamins
  • Digestion
  • Insulation
  • Membrane structure: Hydrophobic properties

Lipid types

lipid types

lipid types

nat occuring FAs in mammals

nat occuring FAs in mammals

Read Full Post »

The multi-step transfer of phosphate bond and hydrogen exchange energy

Curator: Larry H. Bernstein, MD, FCAP, Leaders in Pharmaceutical Intelligence

In this subtext of the series we expand on a tie between respiration and glycolysis, and the functioning of the mitochondrion to discover the key role played by oxidative phosphorylation, “acetyl coenzyme A, and electron transport.  This was crucial to understanding cellular energetics, which explains the high energy of fatty acid catabolism from stored adipose tissue, and the criticality of the multi-step sequence of reactions in energy transfer.

This portion considerably provides a response to the TWO points made by Jose EDS Rosallis:

  1. Just at the beginning, when phosphorylation of proteins is presented, I assume you must mention that some proteins are activated by phosphorylation. This is fundamental in order to present self –organization reflex upon fast regulatory mechanisms. This poiny needs further clarification, but he makes important observations here.
  • Even from an historical point of view. The first observation arrived from a sample due to be studied on the following day of glycogen synthetase. It was unintended left overnight out of the refrigerator. The result was it had changed from active form of the previous day to a non-active form.

The story could have being finished here, if the researcher did not decide to spent this day increasing substrate levels (it could be a simple case of denaturation of proteins that changes its conformation despite the same order of amino acids). He kept on trying and found restoration of maximal activity.

  • This assay was repeated with glycogen phosphorylase and the result was the opposite it increases its activity.

This led to the discovery of cAMP activated protein kinase and the assembly of a very complex system in the glycogen granule that is not a simple carbohydrate polymer. Instead

  • it has several proteins assembled and preserves the capacity to receive from a single event (rise in cAMP) two opposing signals with maximal efficiency,
  • stops glycogen synthesis, as long as levels of glucose 6 phosphate are low and
  • increases glycogen phosphorylation as long as AMP levels are high).

I did everything I was able to do by the end of 1970 in order to repeat these assays with

  • PK I, PKII and PKIII of M. Rouxii and Sutherland route to cAMP failed in this case.

I ask Leloir to suggest to my chief (SP) the idea of AA, AB, BB subunits as was observed in lactic dehydrogenase (tetramer)
(Nathan O. Kaplan discovery) indicating this as his idea. The reason was my “chief” (SP) more than once,  said to me: “Leave these great ideas for the Houssay, Leloir etc…We must do our career with small things. ” However, as she also had a faulty ability for recollection she also used to arrive some time later, with the very same idea but in that case, as her idea.

[This reminds me of when I was studying the emergence of lactic dehysrogenase isoenzyme patterns in the developing eye lens of cattle, I raised reservations about Elliott Vessells challenge to Nathan Kaplan, but that not being my primary problem, my brilliant mentor (H.M.), a very young full professor of anatomy said – leave that to NOK.}

Leloir, said to me: I will not offer your interpretation to her as mine. I think it is not phosphorylation, however I think it is

  • glycosylation that explains the changes in the isoenzymes with the same molecular weight preserved.

This dialogue explains why during the Schroedinger’s “What is life?” reading with him he asked me if from biochemist in exile, to biochemist I expressed all of my thoughts to him. Since I had considered that Schrödinger did not confront Darlington & Haldane for being in exile. This may explain why Leloir could have answered a bad telephone call from P. Boyer, Editor of The Enzymes in a way that suggests the the pattern could be of covalent changes over a protein. Our FEBS and Eur J. Biochemistry papers on pyruvate kinase of M. Rouxii is wrongly quoted in this way on his review about pyruvate kinase of
that year(1971).

  1. show in detail with different colors what carbons belongs to CoA a huge molecule, in comparison with the single two carbons of acetate that will produce the enormous jump in energy yield in comparison with anaerobic glycolysis. The idea is how much must have being spent in DNA sequences to build that molecule in order to use only two atoms of carbon. Very limited aspects of biology could be explained in this way. In case we follow an alternative way of thinking, it becomes clearer that proteins were made more stable by interaction with other molecules (great and small). Afterwards, it rather easy to understand how the stability of protein-RNA complexes where transmitted to RNA (vibrational +solvational reactivity stability pair of conformational energy). Latter, millions of years, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.

The outline of what I am presenting in series is as follows:

  1. Signaling and Signaling Pathways
  1. Signaling transduction tutorial.
  1. Carbohydrate metabolism

3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence

  1. Lipid metabolism

4.1  Studies of respiration lead to Acetyl CoA

4.2 The multi-step transfer of phosphate bond and hydrogen exchange energy

  1. Protein synthesis and degradation
  2. Subcellular structure
  3. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Oxidation-Reduction Reactions

Rachel Casiday, Carolyn Herman, and Regina Frey
Department of Chemistry, Washington University
St. Louis, MO 63130


OX-Phos steps

OX-Phos steps


Key Concepts:

  • ATP as Free-Energy Currency in the Body
  • Coupled Reactions
    • Standard Free-Energy Change for Coupled Reactions
    • ATP Dephosphorylation Coupled to Nonspontaneous Reactions
    • Coupled Reactions to Generate ATP
  • Structure and Function of the Mitochondria
  • Oxidation-Reduction Reactions in the Electron-Transport Chain
    • Electron-Carrier Proteins (NOTE: This section includes a separate link and an animation.)
    • Relationship Between Reduction Potentials and Free Energy
  • Proton Gradient as Means of Coupling Oxidative and Phosphorylation Components of Oxidative Phosphorylation
  • ATP Synthetase Uses Energy From Proton Gradient to Generate ATP

Every day, we build bones, move muscles, eat food, think, and perform many other activities with our bodies. All of these activities are based upon chemical reactions. However, most of these reactions are not spontaneous (i.e., they are accompanied by a positive change in free energy, DG>0) and do not occur without some other source of free energy. Hence, the body needs some sort of “free-energy currency,” (Figure 1) a molecule that can store and release free energy when it is needed to power a given biochemical reaction.

The four questions:

  1. How does the body “spend” free-energy currency to make a nonspontaneous reaction spontaneous? The answer, which is based on thermodynamics, is to use coupled reactions.
  2. How is food used to produce the reducing agents (NADH and FADH2) that can regenerate the free-energy currency? The answer, from biology, is found in glycolysis and the citric-acid cycle.
  3. How are the reducing agents (NADH and FADH2) able to generate the free-energy currency molecule (ATP)? Once again, coupled reactions are key.
  4. What mechanism does the body use to couple the reducing agent reactions and the generation of ATP? ATP is synthesized primarily by a two-step process consisting of an electron-transport chain and a proton gradient.  This process is based on electrochemistry and equilibrium, as well as thermodynamics.

The free-energy change (DG) for the net reaction is given by the sum of the free-energy changes for the individual reactions.  The phospholipids that form cell membranes are formed from glycerol with a phosphate group and two fatty-acid chains attached.This step actually consists of two reactions:

(1) the phosphorylation of glycerol, and

(2) the dephosphorylation of ATP (the free-energy-currency molecule). The reactions may be added as shown in Equations 2-4, below:

      Glycerol + HPO42- –>  (Glycerol-3-Phosphate)2- + H2O DGo= +9.2 kJ
+      ATP4- + H2O –>       ADP3- + HPO42- + H+ DGo30.5 kJ
     Glycerol + ATP4- –> (Glycerol-3-Phosphate)2- +ADP3- + H+ DGo21.3 kJ

ATP is the most important “free-energy-currency” molecule in living organisms (see Figure 2, below). Adenosine triphosphate (ATP) is a useful free-energy currency because the dephosphorylation reaction is very spontaneous; i.e., it releases a large amount of free energy (30.5 kJ/mol). Thus, the dephosphorylation reaction of ATP to ADP and inorganic phosphate (Equation 3) is often coupled with nonspontaneous reactions (e.g., Equation 2) to drive them forward. The body’s use of ATP as a free-energy currency is a very effective strategy to cause vital nonspontaneous reactions to occur.

structure of ATP

structure of ATP

This is the two-dimensional (ChemDraw) structure of ATP, adenosine triphosphate. The removal of one phosphate group (green) from ATP requires the breaking of a bond (blue) and results in a large release of free energy. Removal of this phosphate group (green) results in ADP, adenosine diphosphate.

flowchart of food energy

flowchart of food energy

This flowchart shows that the energy used by the body for its many activities ultimately comes from the chemical energy in our food. The chemical energy in our food is converted to reducing agents (NADH and FADH2). These reducing agents are then used to make ATP. ATP stores chemical energy, so that it is available to the body in a readily accessible form.

Glycolysis   Glucose + 2 HPO42- + 2 ADP3- + 2 NAD+ –>
2 Pyruvate + 2 ATP4- + 2 NADH + 2 H+ + 2 H2O
Intermediate Step   2(Pyruvate + Coenzyme A + NAD+ –>
Acetyl CoA + CO2 + NADH)
Citric-Acid Cycle 2(Acetyl CoA + 3 NAD++ FAD + GDP3-
+ HPO42- + 2H2O –> 2 CO2 + 3 NADH + FADH2
+ GTP4- + 2H+ + Coenzyme A)

The structures of the important molecules in Equations 5-7 are shown in Table 1, below.

How is Food Used to Make the Reducing Agents Needed for the Production of ATP?

To make ATP, energy must be absorbed. This energy is supplied by the food we eat, and then used to synthsize two reducing agents, NADH and FADH2 that are needed to produce ATP. One of the principal energy-yielding nutrients in our diet is glucose (see structure in Table 1 in the blue box below), a simple six-carbon sugar that can be broken down by the body. When the chemical bonds in glucose are broken, free energy is released. The complete breakdown of glucose into CO2 occurs in two processes: glycolysis and the citric-acid cycle. The reactions for these two processes are shown in the blue box below.






Acetyl CoA








two-dimensional representations of several important molecules in Equations 5-7.

As seen in Equations 5-7 in the blue box, glycolysis and the citric-acid cycle produce a net total of only four ATP or GTP molecules (GTP is an energy-currency molecule similar to ATP) per glucose molecule. This yield isfar below the amount needed by the body for normal functioning, and in fact is far below the actual ATP yield for glucose in aerobic organisms (organisms that use molecular oxygen). For each glucose molecule the body processes, the body actually gains approximately 30 ATP molecules! (See Figure 4, below.)  So, how does the body generate ATP?

The process that accounts for the high ATP yield is known as oxidative phosphorylation. A quick examination of Equations 5-7 shows that glycolysis and the citric-acid cycle generate other products besides ATP and GTP, namely NADH and FADH2 (blue). These products are molecules that are oxidized (i.e., give up electrons) spontaneously. The body uses these reducing agents (NADH and FADH2) in an oxidation-reduction reaction .  As you will see later in this tutorial, it is the free energy from these redox reactions that is used to drive the production of ATP.

flowchart - making of ATP

flowchart – making of ATP

This flowchart shows the major steps involved in breaking down glucose from the diet and converting its chemical energy to the chemical energy in the phosphate bonds of ATP, in aerobic (oxygen-using) organisms. Note: In this flowchart, red denotes a source of carbon atoms (originally from glucose),green denotes energy-currency molecules, and blue denotes the reducing agents that can be oxidized spontaneously.

In the discussion above, we see that glucose by itself generates only a tiny amount of ATP. However, during the breakdown of glucose, a large amount of NADH and FADHis produced; it is these reducing agents that dramatically increase the amount of ATP produced. How does this work?

How are the reducing agents (NADH and FADH2) able to generate the free-energy currency molecule (ATP)?

As discussed in an earlier section about coupling reactions, ATP is used as free-energy currency by coupling its (spontaneous) dephosphorylation (Equation 3) with a (nonspontaneous) biochemical reaction to give a net release of free energy (i.e., a net spontaneous reaction). Coupled reactions are also used to generate ATP by phosphorylating ADP. The nonspontaneous reaction of joining ADP to inorganic phosphate to make ATP (Equation 8, below, and Figure 2, above) is coupled to the oxidation reaction of NADH or FADH(Equation 9, below). (Recall, NADH and FADH2 are produced in glycolysis and the citric-acid cycle as described in the blue box). For simplicity, we shall henceforth discuss only the oxidation of NADH; FADH2 follows a very similar oxidation pathway.

The oxidation reaction for NADH has a larger, but negative, DG than the positive DG required for the formation of ATP from ADP and phosphate. This set of coupled reactions is so important that it has been given a special name: oxidative phosphorylation. This name emphasizes the fact that an oxidation (of NADH) reaction (Equation 9 and Figure 5, below) is being coupled to a phosphorylation (of ADP) reaction (Equation 8, below, and Figure 2, above). In addition, we must consider the reduction reaction (gaining of electrons) that accompanies the oxidation of NADH. (Oxidation reactions are always accompanied by reduction reactions, because an electron given up by one group must be accepted by another group.) In this case, molecular oxygen (O2) is the electron acceptor, and the oxygen is reduced to water (Equation 10, below) .

The individual reactions of interest for oxidative phosphorylation are:


ADP3- + HPO42- + H+ –>
ATP4- + H2O

DGo= +30.5 kJ

NADH –> NAD+ + H+ +  2e

DGo158.2 Kj

1/2 O2 + 2H+ + 2e –> H2O

DGo61.9 kJ


The net reaction is obtained by summing the coupled reactions, as shown in Equation 11, below.

ADP3- + HPO42- + NADH + 1/2 O2 + 2H+ –>
ATP4- + NAD+ + 2 H2O
DGo= -189.6 kJ

The molecular changes that occur upon oxidation of NADH are shown:



This is a two-dimensional (ChemDraw) representation showing the change that occurs when NADH is oxidized to NAD+. “R” represents the part of the structure that is shown in black in the drawing of NADH in Table 1, and does not change during the oxidation half-reaction. The molecular changes that occur upon oxidation are shown in red.

In this tutorial, we have seen that nonspontaneous reactions in the body occur by coupling them with a very spontaneous reaction (usually the ATP reaction shown in Equation 3). We have just seen that ATP is produced by coupling the phosphorylation reaction with NADH oxidation (a very spontaneous reaction). But we have not yet answered the question: by what mechanism are these reactions coupled?

Coupling Reactions in Biological Systems

Every day your body carries out many nonspontaneous reactions. As discussed earlier, if a nonspontaneous reaction is coupled to a spontaneous reaction, as long as the sum of the free energies for the two reactions is negative, the coupled reactions will occur spontaneously. How is this coupling achieved in the body? Living systems couple reactions in several ways, but the most common method of coupling reactions is to carry out both reactions on the same enzyme. Consider again the phosphorylation of glycerol (Equations 2-4). Glycerol is phosphorylated by the enzyme glycerol kinase, which is found in your liver. The product of glycerol phosporylation, glycerol-3-phosphate (Equation 2), is used in the synthesis of phospholipids.

Glycerol kinase is a large protein comprised of about 500 amino acids. X-ray crystallography of the protein shows us that there is a deep groove or cleft in the protein where glycerol and ATP attach (see Figure 6, below). Because the enzyme holds the ATP and the glycerol in place, the phosphate can be transferred directly from the ATP to glycerol. Instead of two separate reactions where ATP loses a phosphate (Equation 3) and glycerol picks up a phosphate (Equation 2), the enzyme allows the phosphate to move directly from ATP to glycerol (Equation 4).

The coupling in oxidative phosphorylation uses a more complicated (and amazing!) mechanism, but the end result is the same: the reactions are linked together, the net free energy for the linked reactions is negative, and, therefore, the linked reactions are spontaneous.



This is a schematic representation of ATP and glycerol bound (attached) to glycerol kinase. The enzyme glycerol kinase is a dimer (consists of two identical subuits). There is a deep cleft between the subunits where ATP and glycerol bind. Since the ATP and phosphate are physically so close together when they are bound to the enzyme, the phosphate can be transferred directly from ATP to glycerol. Hence, the processes of ATP losing a phosphate (spontaneous) and glycerol gaining a phosphate (nonspontaneous) are linked together as one spontaneous process

Questions on ATP: The Body’s Free-Energy Currency (How Free-Energy Currency Works)

  • Biological systems involve many molecules containing phosphate groups, such as ATP. Although ATP is the most commonly used free-energy currency, any of these phosphorylated molecules could, in theory, be used as free-energy currency. The standard free-energy change (DGo) for the dephosphorylation (removal of a phosphate group) of several biological compounds is given below:
Acetyl phosphate DGo = -47.3 kJ/mol
Adenosine triphosphate (ATP) DGo = -30.5 kJ/mol
Glucose-6-phosphate DGo = -13.8 kJ/mol
Phosphoenolpyruvate (PEP) DGo = -61.9 kJ/mol
Phosphocreatine DGo = -43.1 kJ/mol

Neglecting any differences in difficulty synthesizing or accessing these molecules by biological systems, rank the molecules in order of their efficiency as a free-energy currency (i.e., the amount of nonspontaneous reactions enabled per phosphate removed from a molecule of free-energy currency) from the most efficient to the least efficient.

  • What, if any, changes are there in the shape of the ring as NADH is oxidized to NAD+(see Figure 5)? (Hint: Consider which atoms lie in the same plane in each structure.)

Mechanism of Coupling the Oxidative-Phosphorylation Reactions

In order to couple the redox and phosphorylation reactions needed for ATP synthesis in the body, there must be some mechanism linking the reactions together. In cells, this is accomplished through an elegant proton-pumping system that occurs inside special double-membrane-bound organelles (specialized cellular components) known as mitochondria. A number of proteins are required to maintain this proton-pumping system and catalyze the oxidative and phosphorylation reactions.

Synthesis of ATP (Equation 8) is coupled with the oxidation of NADH (Equation 9) and the reduction of O2 (Equation 10). There are three key steps in this process:

  1. Electrons are transferred from NADH, through a series of electron carriers, to O2. The electron carriers are proteins embedded in the inner mitochondrial membrane. (More detail about the structure of the mitochondria is presented in the next section.) (See Figure 7a.)
  2. Transfer of electrons by these carriers generates a proton (H+) gradient across the inner mitochondrial membrane. (See Figure 7b.)
  3. When Hspontaneously diffuses back across the inner mitochondrial membrane, ATP is synthesized. The large positive free energy of ATP synthesis is overcome by the even larger negative free energy associated with proton flow down the concentration gradient. (See Figure 7c.)

These steps are outlined below.

  1. Electron Transport (Oxidation-Reduction Reactions) Through a Series of Proteins in the Inner Membrane of the Mitochondria


Generation of H+(Proton) Concentration Gradient Across the Inner Mitochondrial Membrane During the Electron-Transport Process (via a Proton Pump)

. Generation of H+ (Proton) Concentration Gradient Across the Inner Mitochondrial Membrane

. Generation of H+ (Proton) Concentration Gradient Across the Inner Mitochondrial Membrane

Synthesis of ATP Using Free Energy Released From Spontaneous Diffusion of H+Back to the Matrix Inside the Inner Mitochondrial Membrane

. Synthesis of ATP Using Free Energy Released From Spontaneous Diffusion of H+

. Synthesis of ATP Using Free Energy Released From Spontaneous Diffusion of H+

The three major steps in oxidative phosphorylation are

(a) oxidation-reduction reactions involving electron transfers between specialized proteins embedded in the inner mitochondrial membrane; 

(b) the generation of a proton (H+) gradient across the inner mitochondrial membrane (which occurs simultaneously with step (a)); and 

(c) the synthesis of ATP using energy from the spontaneous diffusion of electrons down the proton gradient generated in step (b).

Note: Steps (a) and (b) show cytochrome oxidase, the final electron-carrier protein in the electron-transport chain described above. When this protein accepts an electron (green) from another protein in the electron-transport chain, an Fe(III) ion in the center of a heme group (purple) embedded in the protein is reduced to Fe(II). The coordinates for the protein were determined using x-ray crystallography, and the image was rendered using SwissPDB Viewer and POV-Ray (see References).

Cells use a proton-pumping system made up of proteins inside the mitochondria to generate ATP. Before we examine the details of ATP synthesis, we shall step back and look at the big picture by exploring the structure and function of the mitochondria, where oxidative phosphorylation occurs.

Structure and Function of the Mitochondria



This is a schematic diagram showing the membranes of the mitochondrion. The purple shapes on the inner membrane represent proteins, which are described in the section below. An enlargement of the boxed portion of the inner membrane in this figure is shown in Figure.

The mitochondrial membranes are crucial for this organelle’s role in oxidative phosphorylation. As shown in Figure 8, mitochondria have two membranes, an inner and an outer membrane. The outer membrane ispermeable to most small molecules and ions, because it contains large protein channels called porins. The inner membrane is impermeable to most ions and polar molecules. The inner membrane is the site of oxidative phosphorylation. Although the membrane is mostly impermeable, it contains special H+ (proton) channels and pumps that enable the coupling of the redox reaction involving NADH and O2 (Equations 9-10) to the phosphorylation reaction of ADP (Equation 8), as described below (“Oxidation-Reduction Reactions and Proton Pumping in Oxidative Phosphorylation”). (Recall the discussion of protein channels in the “Maintaining the Body’s Chemistry: Dialysis in the Kidneys” Tutorial .)

As shown in Figure 8, inside the inner membrane is a space known as the matrix; the space between the two membranes is known as the intermembrane space. The matrix side of the inner membrane has a negative electrical charge relative to the intermembrane space due to an H+ gradient set up by the redox reaction (Equations 9 and 10). This charge difference is used to provide free energy (G) for the phosphorylation reaction (Equation 8).

Oxidation-Reduction Reactions and Proton Pumping in Oxidative Phosphorylation

Phosphorylation of ADP (Equation 8) is coupled to the oxidation-reduction reaction of NADH and O2 (Equations 9 and 10). Electrons are not transferred directly from NADH to O2, but rather pass through a series of intermediate electron carriers in the inner membrane of the mitochondrion. Why? This allows something very important to occur: the pumping of protons across the inner membrane of the mitochondrion. As we shall see, it is this proton pumping that is ultimately responsible for coupling the oxidation-reduction reaction to ATP synthesis.

Two major types of mitochondrial proteins (see Figure 9, below) are required for oxidative phosphorylation to occur. Both classes of proteins are located in the inner mitochondrial membrane.

  1. The electron carriers (NADH-Q reductase, ubiquinone (Q), cytochrome reductase, cytochrome c, and cytochrome oxidase shown in shades of purple in Figure 9 below) transport electrons in a stepwise fashion from NADH to O2.  Three of these carriers (NADH-Q reductase, cytochrome reductase, and cytochrome oxidase) are also proton pumps, and simultaneously pump H+ ions (protons) from the matrix to the intermembrane space. (Proton movement from one side of the membrane to the other is shown as blue arrows in Figure 9, below.) The protons that are pumped across the membrane complete the redox reaction (Equations 9 and 10). The creation of a proton gradient across the membrane is one way of storing free energy.
  2. ATP synthetase (shown in red in Figure 9 below) allows H+ ions to diffuse back into the matrix and uses the free energy released to synthesize ATP from ADP and HPO42-. The ATP synthetase is essential for the phosphorylation to occur (Equation 8). (Proton movement from one side of the membrane to the other is shown as blue arrows in Figure 9, below.)

The electron carriers can be divided into three protein complexes (NADH-Q reductase (1), cytochrome reductase (3), and cytochrome oxidase (5)) that pump protons from the matrix to the intermembrane space, and two mobile carriers (ubiquinone (2) and cytochrome c (4)) that transfer electrons between the three proton-pumping complexes. (Gold numbers refer to the labels on each protein in Figure 9, below.) Because electrons move from one carrier to another until they are finally transferred to O2, the electron carriers (shown in Figure 9,below) are said to form an electron-transport chain.

Figure  below, is a schematic representation of the proteins involved in oxidative phosphorylation. To see an animation of oxidative phosphorylation, click on “View the Movie.”

Proteins of inner space

Proteins of inner space

This is a schematic diagram illustrating the transfer of electrons from NADH, through the electron carriers in the electron transport chain, to molecular oxygen. Please click on the pink button below to view a QuickTime animation of the functions of the proteins embedded in the inner mitochondrial membrane that are necessary for oxidative phosphorylation. Click the blue button below to download QuickTime 4.0 to view the movie.

NADH-Q reductase (1), cytochrome reductase (3) , and cytochrome oxidase (5) are electron carriers as well as proton pumps, using the energy gained from each electron-transfer step to move protons (H+) against a concentration gradient, from the matrix to the intermembrane space.Ubiquinone (Q) (2) and cytochrome c (Cyt C) (4) are mobile electron carriers. (Ubiquinone is not actually a protein.) All of the electron carriers are shown in purple, with lighter shades representing increasingly higher reduction potentials. Together, these electron carriers form a “chain” to transport electrons from NADH to O2. The path of the electrons is shown with the green dotted line.

ATP synthetase (red) has two components: a proton channel (allowing diffusion of protons down a concentration gradient, from the intermembrane space to the matrix), and a catalytic component to catalyze the formation of ATP.

For a more complete description of each step in oxidative phosphorylation (indicated by the gold numbers), click here.

view movie

view movie

Click here for a brief description of each of the electron carriers in the electron-transport chain. It is important to note that, although NADH donates two electrons and O2 ultimately accepts four electrons, each of the carriers can only transfer one electron at a time. Hence, there are several points along the chain where electrons can be collected and dispersed. For the sake of simplicity, these points are not described in this tutorial.

In the section above, we see that the oxidation-reduction process is a series of electron transfers that occurs spontaneously and produces a proton gradient. Why are the electron tranfers from one electron carrier to the next spontaneous?

What causes electrons to be transferred down the electron-transport chain?

As seen in Table 2, below, and Figure 7a, in these carriers, the species being oxidized or reduced is Fe, which is found either in a iron-sulfur (Fe-S) group or in a heme group. (Recall the heme group from the Chem 151 tutorial “Hemoglobin and the Heme Group: Metal Complexes in the Blood“.) The iron in these groups is alternately oxidized and reduced between Fe(II) (reduced) or Fe(III) (oxidized) states.

Table 2 shows that the electrons are transferred through the electron-transport chain because of the difference in the reduction potential of the electron carriers. As explained in the green box below, the higher the electrical potential (e) of a reduction half reaction is, the greater the tendency is for the species to accept an electron. Hence, in the electron-transport chain, electrons are transferred spontaneously from carriers whose reduction results in a small electrical potential change to carriers whose reduction results in an increasingly larger electrical potential change.

Reduction Potentials and Relationship to Free Energy

An oxidation-reduction reaction consists of an oxidation half reaction and a reduction half reaction. Every half reaction has an electrical potential (e). By convention, all half reactions are written as reductions, and the electrical potential for an oxidation half-reaction is equal in magnitude, but opposite in sign, to the electrical potential for the corresponding reduction (i.e., the opposite reaction). The electrical potential for an oxidation-reduction reaction is calculated by

erxn = eoxidation + ereduction (12)

For example, for the overall reaction of the oxidation of NADH paired with the reduction of O2, the potential can be calculated as shown below.

Reduction Potentials ereduction
NAD+ + 2H+ + 2e –> NADH + H+ -0.32 V
(1/2) O2 + 2H+ + 2e –> H2O +0.82 V

The overall reaction is

NADH + H–> NAD+ + 2H+ + 2e eoxidation = 0.32 V
(1/2) O2 + 2H+ + 2e –> H2O ereduction = 0.82 V
net: NADH + (1/2)O2 + H+ –>
H2O + NAD+
erxn = 1.14 V

The electrical potential (erxn) is related to the free energy (DG) by the following equation:

DG= -nFerxn (13)

where n is the number of electrons transferred (in moles, from the balanced equation), and F is the Faraday constant (96,485 Coulombs/mole). (Using this equation, DG is given in Joules; one Joule = 1 Volt x 1 Coulomb.)

Hence the overall reaction for the oxidation of NADH paired with the reduction of O2 has a negative change in free energy (DG =-220 kJ); i.e., it is spontaneous. Thus, the higher the electrical potential of a reduction half reaction, the greater the tendency for the species to accept an electron.

Just as in the box above, the electrical potential for the overall reaction (electron transfer) between two electron carriers is the sum of the potentials for the two half reactions. As long as the potential for the overall reaction is positive the reaction is spontaneous. Hence, from Table 2 below, we see that cytochrome c1 (part of the cytochrome reductase complex, #3 in Figure 9) can spontaneously transfer an electron to cytochrome c (#4 in Figure 9). The net reaction is given by Equation 16, below.

reduced cytochrome c–> oxidized cytochrome c+ e eoxidation = – .220 V (14)
oxidized cytochrome c + e –> reduced cytochrome c ereduction = .250 V (15)
NET: reduced cyt c1 + oxidized cyt c –>
oxidized cyt c+ reduced cyt c
erxn = 0.030 V (16) Spontaneous

We can also see from Table 2 that cytochrome c1 cannot spontaneously transfer an electron to cytochrome b (Equation 19):

reduced cyt c–> oxidized cyt c+ e eoxidation = – .220 V (17)
oxidized cyt b + e –> reduced cyt b ereduction = – 0.34 V (18)
NET: reduced cyt c1 + oxidized cyt c –>
oxidized cyt c+ reduced cyt c
erxn = – 0.56 V (19) NOT Spontaneous

Table 2 lists the reduction potentials for each of the cytochrome proteins (i.e., the last three steps in the electron-transport chain before the electrons are accepted by O2) involved in the electron-transport chain. Note that each electron transfer is to a cytochrome with a higher reduction potential than the previous cytochrome. As described in the box above and seen in Equations 14-19, an increase in potential leads to a decrease in DG (Equation 13), and thus the transfer of electrons through the chain is spontaneous.

Complex Name Half Reaction Reduction Potential
Cytochrome reductase

(also known as cytochrome b-c1 complex)

(3 in Figure 9)

Cytochrome b (Fe(III) center)
+ e –>
Cytochrome b (Fe(II) center)
-0.34 V
(at pH 7, T=30oC)
Cytochrome c1 (Fe(III) center)
+ e– –>
Cytochrome c1 (Fe(II) center)
+0.220 V
(at pH 7, T=30oC)
Cytochrome c

(4 in Figure 9)

Cytochrome c (Fe(III) center)
+ e– –>
Cytochrome c (Fe(II) center)
+0.250 V
(at pH 7, T=30oC)
Cytochrome oxidase

(5 in Figure 9)

Cytochrome oxidase
( Fe(III) center) + e– –>
Cytochrome oxidase
(Fe(II) center)
+0.285 V
(at pH 7.4, T=25oC)
Table 2

To view the cytochrome molecules interactively using RASMOL, please click on the name of the complex to download the pdb file.

Hence, the electron-transport chain (which works because of the difference in reduction potentials) leads to a large concentration gradient for H+. As we shall see below, this huge concentration gradient leads to the production of ATP.

Questions on Electron Carriers: Steps in the Electron-Transport Chain; Reduction Potentials and Relationship to Free Energy

  • Briefly, explain why electrons travel from NADH-Q reductase, to ubiquinone (Q), to cytochrome reductase, rather than in the opposite direction.
  • One result of the transfer of electrons from NADH-Q reductase down the electron transport chain is that the concentration of protons (H+ ions) in the intermembrane space is increased.  Could cells move protons (H+ ions) from the matrix to the intermembrane space without transporting electrons?  Why or why not?

 ATP Synthetase: Production of ATP

We have seen that the electron-transport chain generates a large proton gradient across the inner mitochondrial membrane. But recall that the ultimate goal of oxidative phosphorylation is to generate ATP to supply readily-available free energy for the body. How does this occur? In addition to the electron-carrier proteins embedded in the inner mitochondrial membrane, a special protein called ATP synthetase (Figure 9, the red-colored protein) is also embedded in this membrane. ATP synthetase uses the proton gradient created by the electron-transport chain to drive the phosphorylation reaction that generates ATP (Figure 7c).

ATP synthetase is a protein consisting of two important segments: a transmembrane proton channel, and a catalytic component located inside the matrix. The proton-channel segment allows H+ ions to diffuse from the intermembrane space, where the concentration is high, to the matrix, where the concentration is low. Recall from the Kidney Dialysis tutorial that particles spontaneously diffuse from areas of high concentration to areas of low concentration. Thus, since the diffusion of protons through the channel component of ATP synthetase is spontaneous, this process is accompanied by a negative change in free energy (i.e., free energy is released). The catalytic component of ATP synthetase has a site where ADP can enter. Then, using the free energy released by the spontaneous diffusion of protons through the channel segment, a bond is formed between the ADP and a free phosphate group, creating an ATP molecule. The ATP is then released from the reaction site, and a new ADP molecule can enter in order to be phosphorylated.

Questions on ATP Synthetase: Production of ATP

  • A scientist has created a phospholipid-bilayer membrane containing ATP-synthetase proteins. Instead of a proton gradient, this scientist has created a large Cs+ gradient (many Cs+ ions on the side of the membrane without the catalytic unit, and few Cs+ ions on the side of the membrane with the catalytic unit). Would you expect the ATP-synthetase proteins in this membrane to be able to generate ATP, given an abundant supply of ADP and phosphate? Briefly, explain your answer. (HINT: Draw on your knowledge of the structure of protein channels to predict what effect replacing H+ ions with Cs+ ions would have.)
  • Certain toxins allow H+ ions to move freely across the inner mitochondrial membrane (i.e., without needing to pass through the channel in ATP synthetase). What effect do you expect these toxins to have on the production of ATP? Briefly, explain your answer.


In this tutorial, we have learned that the ability of the body to perform daily activities is dependent on thermodynamic, equilibrium, and electrochemical concepts.   These activities, which are typically based on nonspontaneous chemical reactions, are performed by using free-energy currency. The common free-energy currency is ATP, which is a molecule that easily dephosphorylates (loses a phosphate group) and releases a large amount of free energy. In the body, the nonspontaneous reactions are coupled to this very spontaneous dephosphorylation reaction, thereby making the overall reaction spontaneous (DG < 0). As the coupled reactions occur (i.e., as the body performs daily activities), ATP is consumed and the body regenerates ATP by using energy from the food we eat (Figure 3). As seen in Figure 4, the breakdown of glucose (glycolysis) obtained from the food we eat cannot by itself generate the large amount of ATP that is needed for metabolic energy by the body. However, glycolysis and the subsequent step, the citric-acid cycle, produce two easily oxidized molecules: NADH and FADH2. These redox molecules are used in an oxidative-phosphorylation process to produce the majority of the ATP that the body uses. This oxidative-phosphorylation process consists of two steps: the oxidation of NADH (or FADH2) and the phosphorylation reaction which regenerates ATP. Oxidative phosphorylation occurs in the mitochondria, and the two reactions (oxidation of NADH or FADHand phosphorylation to generate ATP) are coupled by a proton gradient across the inner membrane of the mitochondria (Figure 9). As seen in Figures 7 and 9, the oxidation of NADH occurs by electron transport through a series of protein complexes located in the inner membrane of the mitochondria. This electron transport is very spontaneous and creates the proton gradient that is necessary to then drive the phosphorylation reaction that generates the ATP. Hence, oxidative-phosphorylation demonstrates that free energy can be easily transferred by proton gradients. Oxidative-phosphorylation is the primary means of generating free-energy currency for aerobic organisms, and as such is one of the most important subjects in the study of bioenergetics (the study of energy and its chemical changes in the biological world).

Additional Link:

  • This fun description of oxidative phosphorylation by Dr. E.J.Oakeley contains step-by-step animated illustrations of the redox reactions involved, as well as a quiz to test your understanding of the material.


Alberts, B. et al. In Molecular Biology of the Cell, 3rd ed., Garland Publishing, Inc.: New York, 1994, pp. 653-684.

Becker, W.M. and Deamer, D.W. In The World of the Cell, 2nd ed., The Benjamin/Cummings Publishing Co., Inc.: Redwood City, CA, 1991, pp. 291-307.

Fasman, G.D. In Handbook of Biochemistry and Molecular Biology, 3rd ed., CRC Press, Inc.: Cleveland, OH, 1976, Vol. I (Physical and Chemical Data), pp. 132-137.

Guex, N. and Peitsch, M.C. Electrophoresis, 1997, 18, 2714-2723. (SwissPDB Viewer) URL:

Moa, C., Ozer, Z., Zhou, M. and Uckun, F. X-Ray Structure of Glycerol Kinase Complexed with an ATP Analog Implies a Novel Mechanism for the ATP-Dependent Gylcerol Phosphorylation by Glycerol Kinase.Biochemical and Biophysical Reaearch Communications. 1999, 259, 640-644.

Persistence of Vision Ray Tracer (POV-Ray). URL:

Stryer, L. In Biochemistry, 4th. ed., W.H. Freeman and Co.: New York, 1995, pp. 490, 509, 513, 529-557.

Zubay, G. Biochemistry, 3rd. ed., Wm. C. Brown Publishers: Dubuque, IA, 1983, p. 42.


The authors thank Dewey Holten (Washington University in St. Louis) for many helpful suggestions in the writing of this tutorial.

The development of this tutorial was supported by a grant from the Howard Hughes Medical Institute, through the Undergraduate Biological Sciences Education program, Grant HHMI# 71199-502008 to Washington University.

Copyright 1999, Washington University, All Rights Reserved.




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Studies of Respiration Lead to Acetyl CoA

Curator: Larry H. Bernstein, MD, FCAP

In this series of discussions it has become clear that the studies of carbohydrate metabolism were highlighted by Meyerhof’s work on the glycolytic pathway, and the further elucidation of a tie between Warburg’s studies of impaired respiration for malignant aerobic cells relying on glycolysis, comparanle to Pasteur’s observations 60 years earlier by for yeast.   The mitochondrion was unknown at the time, and it took many years to discover the key role played by oxidative phosphorylation and Fritz Lipmann’s discovery of “acetyl coenzyme A, and the later explanation of electron transport.  This was crucial to understanding cellular energetics, which explains the high energy of fatty acid catabolism from stored adipose tissue.  I shall here embark on a journey to trace these important connected developments.

  1. Signaling and signaling pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism

3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence

  1. Lipid metabolism

4.1  Studies of respiration lead to Acetyl CoA

  1. Protein synthesis and degradation
  2. Subcellular structure
  3. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.


In some reactions, the purpose of phosphorylation is to “activate” or “volatize” a molecule, increasing its energy so it is able to participate in a subsequent reaction with a negative free-energy change. All kinases require a divalent metal ion such as Mg2+ or Mn2+ to be present, which stabilizes the high-energy bonds of the donor molecule (usually ATP or ATP derivative) and allows phosphorylation to occur.This is a major focus of this discussion.

In other reactions, phosphorylation of a protein substrate can inhibit its activity (as when AKT phosphorylates the enzyme GSK-3). When src is phosphorylated on a particular tyrosine, it folds on itself, and thus masks its own kinase domain, and is thus turned “off”. In still other reactions, phosphorylation of a protein causes it to be bound to other proteins which have “recognition domains” for a phosphorylated tyrosine, serine, or threonine motif. In the late 1990s it was recognized that phosphorylation of some proteins causes them to be degraded by the ATP-dependent ubiquitin/proteasome pathway. This is all that needs to be said at this time about proteins.


Oxidative Phosphorylation

ATP is the molecule that supplies energy to metabolism. Almost all aerobic organisms carry out oxidative phosphorylation. This pathway is probably so pervasive because it is a highly efficient way of releasing energy, compared to alternative fermentation processes such as anaerobic glycolysis.

During oxidative phosphorylation, electrons are transferred from electron donors to electron acceptors such as oxygen, in redox reactions. These redox reactions release energy, which is used to form ATP. In eukaryotes, these redox reactions are carried out by a series of protein complexes within the cell’s intermembrane wall mitochondria, whereas, in prokaryotes, these proteins are located in the cells’ intermembrane space.

O t to  W a r b u r g
Nobel Lecture, December 10, 1931

The oxygen-transferring ferment of respiration

The effects of iron are very great, and it follows that oxidation and reduction of the ferment iron must occur extremely rapidly. In fact, almost every molecule of oxygen that comes into contact with an atom of ferment iron reacts with it.  Complex-bound bivalent iron in compounds reacts, in vitro as well as in the cell, with molecular oxygen. tt is not yet possible to reduce in vitro trivalent iron with the cell fuel: it is always necessary to add a substance of unknown composition, a ferment, that activates the combustible material for the attack of the iron. It must, therefore, be concluded that activation of the combustible substance in the breathing cell precedes the attack of the ferment iron; this corresponds with “hydrogen activation” as postulated in the theory of Wieland and Thunberg. According to the results of a joint research with W. Christian, this is a cleavage comparable with those known as fermentation.

It is possible that the interplay of splitting ferment and oxygen-transferring ferment does not fully explain the mechanism of cellular respiration; that the iron that reacts with the molecular oxygen does not directly oxidize the activated combustible substances, but that it exerts its effects indirectly through still other iron compounds – the three non-auto-oxidizable cell hemes of MacMunn, which occur in living cells according to the spectroscopic observations of MacMunn and Keilin, and which are reduced in the cell under exclusion of oxygen. It is still not possible to answer the question whether the MacMunn hemes form part of the normal respiratory cycle, i.e., whether respiration is not a simple iron catalysis but a four-fold one. The available spectroscopic observations are also consistent with the view that the MacMunn hemes in the cell are only reduced when the concentration of activated combustible substance is physiologically above normal. This will suffice to indicate that oxygen transfer by the iron of the oxygen transferring ferment is not the whole story of respiration. Respiration requires not only oxygen-transferring ferment and combustible substance, but oxygen-transferring ferment and the living cell.

Inhibition of cellular respiration by prussic acid was discovered some 50 years ago by Claude Bernard, and has interested both chemists and biologists ever since. It takes place as the result of a reaction between the prussic acid and the oxygen-transferring ferment iron, that is, with the ferment iron in trivalent form. [In the prussic acid reaction] the oxidizing OH-group of the trivalent ferment-iron is replaced by the non-oxidizing CN-group, thus bringing transfer of oxygen to a standstill. Prussic acid inhibits reduction of the ferment iron. Inhibition of respiration by carbon monoxide was discovered only a few years ago. [Given] the initial reaction in respiration, then, in the presence of carbon monoxide, the competing reaction will also occur and, varying with the pressures of the carbon monoxide and of the oxygen, more or less of the ferment iron will be removed from the catalytic process on account of fixation of carbon monoxide to the ferment iron. Unlike prussic acid, therefore, carbon monoxide affects the bivalent iron of the ferment. Carbon monoxide inhibits oxidation of the ferment iron.

Thus inhibition of respiration by carbon monoxide, unlike that by prussic acid, depends upon the partial pressure of oxygen. The toxic action of prussic acid in the human subject is based on its inhibitory action on cellular respiration. The toxic effect of carbon monoxide on man has nothing to do with inhibition of cellular respiration by carbon monoxide but is based on the reaction of carbon monoxide with blood iron. For, the effect of carbon monoxide on blood iron occurs at pressures of carbon monoxide far from the level at which cellular respiration would be inhibited.

If carbon monoxide is added to the oxygen in which living cells breathe, respiration ceases, as has already been mentioned, but if exposure to ultraviolet or visible light is administered, respiration recurs. By alternate illumination and darkness it is possible to cause respiration and cessation of respiration in living, breathing cells in mixtures of carbon monoxide and oxygen. In the dark, the iron of the oxygen-transferring ferment becomes bound to carbon monoxide, whereas in the light the carbon monoxide is split off from the iron which is, thus, liberated for oxygen transfer. This fact was discovered in 1926 in collaboration with Fritz Kubowitz. Photochemical dissociation of iron carbonyl compounds was discovered in 1891 by Mond and Langer, by exposing iron pentacarbonyl. This reaction is specific for carbonyl compounds of iron, most of which appear to dissociate in the presence of light, e.g., carbon-monoxide hemoglobin (John Haldane, 1897) carbon-monoxide hemochromogen (Anson and Mirsky, 1925), carbon-monoxide pyridine hemochromogen (H. A. Krebs, 1928), and carbon-monoxide ferrocysteine (W. Cremer, 1929).

When the photochemical dissociation of iron carbonyl compounds is measured quantitatively (we followed hereby Emil Warburg’s photochemical experiments), by using monochromatic light and comparing the amount of light energy absorbed with the amount of carbon monoxide set free, it is found that Einstein’s law of photochemical equivalence is very exactly fulfilled. The number of FeCO-groups set free is equal to the number of light quanta absorbed, and this is independent of the wavelength employed.

Photochemical dissociation of iron carbonyl compounds can be used to determine the absorption spectrum of a catalytic oxygen-transferring iron compound. One combines the catalyst in the dark with carbon monoxide, and so abolishes the oxygen-transferring power of the iron. If then this is exposed to monochromatic light of various wavelengths and of measured quantum intensity, and the effect of light W measured the increase in the rate of catalysis – it is found that the effects of the light are proportional to the quanta absorbed. The arrangement becomes very simple if the catalyst is present, as is usually the case, in infinitesimally low concentration in the exposed system. Then the thickness of the layers related to the amount of absorption of light can be considered to be infinitely thin, the number of quanta absorbed is proportional to the number of quanta supplied by irradiation.

In collaboration with Erwin Negelein, this principle was employed to measure the relative absorption spectrum of the oxygen-transferring respiratory ferment. The respiration of living cells was inhibited by carbon monoxide which was mixed with the oxygen. We then irradiated with monochromatic light of various wavelengths and of measured quantum intensity, and [measured] the increase of respiration together with the relative absorption spectrum. Only practically colorless cells are suitable for this type of experiment, [which requires] a layer infinitely thin with regard to light absorption.

Imagine living cells whose respiration is inhibited by carbon monoxide. If these are irradiated, respiration does not increase suddenly from the dark to the light-value, but there is a definite, although very short, interval until the combination of carbon monoxide with the ferment is broken down by the light. Even without calculation, it is obvious that the rate of increase in the effect of light must be related to the depth of colour of the ferment. If the ferment absorbs strongly, the -monoxide compound will be rapidly broken down, and vice versa.

The time of increase of the action of light can be measured. The time taken for a given intensity of light to cause dissociation of approximately half the carbon-monoxide compound of the ferment can be measured and, from this time, and from the effective intensity of light, the absolute absorption coefficient of the ferment for every wavelength can be calculated. The absorption capacity of the ferment, measured in accordance with this principle, was found to be of the same order as the power of light absorption of our strongest pigments. If one imagines a ferment solution of molar concentration, a layer of 2 x 10-6 cm thickness would weaken the blue mercury line 436 µµ up by half. The fact that the ferment in spite of this cannot be seen in the cells is due to its low concentration.

Monochromators and color filters were used to isolate the lines from these sources of light. If the absorption coefficient is entered as a function of the wavelength, the absorption spectrum of the carbon-monoxide compound of the ferment is obtained. The principal absorption-band or y-band lies in the blue.
This is the spectrum of a heme compound, according to the position of the bands, the intensity state of the bands, and the absolute magnitude of the absorption coefficients.

It appeared essential to have a control to ascertain whether heme as an oxidation catalyst of carbon monoxide and prussic acid really behaves like the ferment. If cysteine is dissolved in water containing pyridine, and a trace of heme is added, and this is shaken with air, the cysteine is catalytically oxidized by the oxygen-transferring power of the heme. According to Krebs, the catalysis is inhibited by carbon monoxide in the dark, but the inhibition ceases when the mixture is illuminated. Prussic acid too acts on this model on cellular respiration, inasmuch as it combines with the trivalent heme and inhibits its reduction. Just as in life, inhibition by carbon monoxide is dependent on the oxygen pressure, while inhibition by prussic acid is independent of the oxygen pressure.

In conjunction with Negelein, this model was also used to test the ferment experiments quantitatively. Heme catalysis in the model was inhibited by carbon monoxide in the dark. Then monochromatic light of known quantum intensity was used to irradiate it, and the absorption spectrum of the catalyst calculated from the effect of the light which was known from direct measurements on the pure substance. The calculation gave the absorption spectrum of the heme that had been added as a catalyst, and so the method was verified as a technique for the determination of the ferment spectrum, both the calculation and the measurement method.

The positions of the principal band and a-band of the ferment are:

Principal band            α-band

433 µµ                    590 µµ

These will be referred to as the “ferment bands” because the ferment was the first for which they were determined. Hemes are the complex iron compounds of the porphyrins, in which two valencies of the iron are bound to nitrogen. The porphyrins, of which Hans Fischer determined the chemical structure, are tetrapyrrole compounds in which the four pyrrole nuclei are held together by four interposed methane groups in the cr-position. Green, red, and mixed shades of hemes are known. If magnesium is replaced by iron in chlorophyll, green hemes are obtained. Their color is due to a strong band in the red which is already recognized in chlorophyll. The ferment does not absorb in the red and cannot, therefore, be a green heme. Red hemes are the usual hemes in blood pigment and in its related substances, such as mesoheme and deuteroheme. Coproheme is also a red heme which is an iron compound of the coproporphyrin that H. Fischer recognized in the body. Other red hemes are 20 µµ further from the red than the ferment bands. It follows that the ferment is not a red heme.

The pheoporphyrins are closely related to blood pigment but, as H. Fischer showed, pheoporphyrin a is simply mesoporphyrin in which the one propionic acid has been oxidized so that ring closure with the porphyrin nucleus is made possible. Pheoporphyrin a is a reduction product of chlorophyll a or an oxidation product of blood pigment, and connects together, in an amazingly simple manner, the principal pigments of the organic world the blood pigment and the leaf pigment.

Chlorophyll b has, in general, bands of longer wavelength than chlorophyll a, and for this reason,

  1. Christian and I applied Fischer’s reduction method to it. In this way we obtained pheoheme b, which, when linked with protein, corresponds with the ferment in respect to the position of the principal band. The principal band of the carbon-monoxide compound of pheohemoglobin b is 435 µµ.
  2. However, while the principal band of pheohemoglobin corresponds with the ferment bands within the permitted limits, the α-band shifts so far beyond them because it lies too near the red. It is, nevertheless, interesting that
  3. when ‘chlorophyll b is reduced, one obtains a pheoporphyrin of which the heme of all the pheohemes that have been demonstrated up to the present time is the most like the ferment.


Still nearer the ferment in its spectrum, is a heme occurring in Nature. This is

  • spirographis heme, which has been isolated from chlorocruorin, the blood pigment of the bristle-worm Spirographis,

in collaboration with Negelein and Haas, the bands of spirographis heme, coupled to globin, are :

  • carbon-monoxyhemoglobin of spirographis:  principle band, 434 µµ; α-band, 594 µµ.

Spirographis heme differs from the red hemes by the surplus or ketone oxygen-atom, and is classified as pheoheme. Like Fischer’s pheohemes, spirographis heme is intermediate between chlorophyll and blood pigment in respect of

  • the degree of oxidation of the side-chains.

The two hemes with a spectrum most like that of the ferment – pheoheme b and spirographis heme – possess a remarkable property. If they are dissolved in dilute sodium-hydroxide solution, in the form of ferrous compounds,

  • the absorption bands slowly wander towards the blue, near the bands of blood heme. In this way,
  • mixed-color hemes have been converted into red hemes.

On acidification, the change reverts: the <<blood bands>> disappear and

  • the ferment bands appear.

This experiment shows that

  1. oxidation of the side-chains does not suffice to give rise to the ferment bands, but
  2. some process of the type of anhydride formation must also occur.

The unique intermediate status of the ferment-like hemes demonstrated by these simple experiments suggests

  1. the suspicion that blood pigment and leaf pigments have both arisen from the ferment –
  2. blood pigments by reduction, and leaf pigment by oxidation.
  • For evidently, the ferment existed earlier than hemoglobin and chlorophyll.

The investigations on the oxygen-transferring ferment have been supported from the start by the Notgemeinschaft der deutschen Wissenschaft and the Rockefeller Foundation, without whose help they could not have been carried out. I have to thank both organizations here.

A L B E R T S Z E N T- GY Ö R G Y I      Nobel Lecture, December 11, 1937

Oxidation, energy transfer, and vitamins

A living cell requires energy not only for all its functions, but also

  • for the maintenance of its structure.
  • The source of this energy is the sun’s radiation.

Energy from the sun’s rays is trapped by green plants, and

  • converted into a bound form, invested in a chemical reaction.

When sunlight falls on green-plants, they liberate oxygen from carbon dioxide, and

  1. store up carbon, bound to the elements of water, as carbohydrate.

The radiant energy is now locked up in this carbohydrate molecule. This molecule is our food. When energy is required,

  • the carbohydrate is again combined with oxygen to form carbon dioxide, oxidized, and energy released.

Investigations during the last few decades have brought hydrogen instead of carbon, and instead of CO2 water, the mother of all life, into the foreground. It is becoming increasingly probable that

  1. radiant energy is used primarily to break water down into its elements,
  2. while CO2, serves only to fix the elusive hydrogen thus released.

While this concept of energy fixation was still being developed, the importance of hydrogen in the reversal of this process, whereby energy is liberated by oxidation, had already been confirmed by H. Wieland’s experiments.

Our body really only knowns one fuel, hydrogen. The foodstuff, carbohydrate, is essentially a packet of hydrogen, a hydrogen supplier, a hydrogen donor, and the main event during its combustion is

  • the splitting off of hydrogen.

So the combustion of hydrogen is

  • the real energy-supplying reaction;

To the elucidation of reaction (6), which seems so simple, I have devoted all my energy for the last fifteen years.

When I first ventured into this territory, the foundations had already been laid by the two pioneers H. Wieland and
O. Warburg, and Wieland’s teaching had been applied by Th. Thunberg to the realm of animal physiology.Wieland and Thunberg showed, with regard to foodstuffs, how

  1. the first step in oxidation is the “activation” of hydrogen, whereby
  2. the bonds linking it to the food molecule are loosened, and
  3. hydrogen prepared for splitting off.

But at the same time oxygen is also, as Warburg showed,

  • activated for the reaction by an enzyme.
  • the hydrogen-activating enzymes are called dehydrases or dehydrogenases.

Warburg called his oxygen-activating catalyst, “respiratory enzyme”.These concepts of Wieland and Warburg were apparently contradictory, and

  1. my first task was to show that the two processes are complementary to one another, and that
  2. in muscle cells activated oxygen oxidizes activated hydrogen.

This picture was enriched by the English worker D. Keilin, who showed that

  • activated oxygen does not oxidize activated hydrogen directly, but
  • that a dye, cytochrome, is interposed between them.

In keeping with this function, the “respiratory enzyme” is now also called “cytochrome oxidase”.

About ten years ago, when I tried to construct this system of respiration artificially and added together the respiratory enzyme with cytochrome and some foodstuff together with its dehydrogenase, I could justifiably expect that this system would use up oxygen and oxidize the food. But the system remained inactive. I found that

  • the dehydrogenation of certain donors is linked to the presence of a co-enzyme.

Analysis of this co-enzyme showed it to be a nucleotide, identical with v. Euler’s co-zymase, which H. v. Euler and R. Nilsson had already shown to accelerate the process of dehydration. As a result of Warburg’s investigations,this co-dehydrogenase has recently come very much into the foreground. Warburg showed that

  • it contains a pyridine base, and that it accepts hydrogen directly
    [pyridine nucleotide, triphosphopyridine nucleotide, TPN]

from food when the latter is dehydrogenated. It is therefore, the primary H-acceptor.

While working on the isolation of the co-enzyme with Banga, I found a remarkable dye, which showed clearly by its reversible oxidation that it, too, played a part in the respiration. We called this new dye cytoflav. Later Warburg showed that

  • this substance exercised its function in combination with a protein.

He called this protein complex of the dye, “yellow enzyme”. R. Kuhn, to whom we owe the structural analysis of the dye, called the dye lactoflavin and, with Györgyi and Wagner-Jauregg, showed it to be identical with vitamin B,.But the respiratory system stayed inactive even

  • after the addition of both these new components, codehydrogenase and yellow enzyme.

The C4-dicarboxylic acids and their activators which Thunberg discovered are

  • interposed between cytochrome and the activation of hydrogen as intermediate hydrogen-carriers.

In the case of carbohydrate, hydrogen from the food is first taken up by oxaloacetic acid, which

  • is reacted with the cytoplasmic malic dehydrogenase (and pyridine nucleotide –
    reduced DPN[H])
    , and thereby activated.

By taking up two hydrogen atoms, oxaloacetic acid is changed into malic acid.

  • OAA + NADH – (MDH) – malate + NAD+ + H+

This malic acid now passes on the H-atoms, and thus reverts to oxaloacetic acid,

  • which can again take up new H-atoms.

Malate + NAD+ + H+ — MDH – OAA + NADH

The H-atoms released by malic acid are taken up by fumaric acid, which is similarly

  • activated by the so-called succinic dehydrogenase.

The uptake of two H-atoms

  • converts the fumarate to succinate, to succinic acid.

The two H-atoms of succinic acid are then

  • oxidized away by the cytochrome.

Finally the cytochrome is oxidized by the respiratory enzyme, and

  • the respiratory enzyme by oxygen.

The function of the C4-dicarboxylic acids is not to be pictured as consisting of a certain amount of C4-dicarboxylic acid in the cell which is alternately oxidized and reduced. Fig. 2 corresponds more to the real situation. The protoplasmic surface, which is represented by the semi-circle, has single molecules of oxaloacetate and fumarate attached to it as prosthetic groups. These fused, activated dicarboxylic molecules then temporarily bind the hydrogen from the food. The co-dehydrogenases and the yellow enzymes also take part in this system. I have attempted to add them in at the right place.

This diagram, which will probably still undergo many more modifications, states that the “foodstuff” – H-donor – starts by

  1. passing its hydrogen, which has been activated by dehydrase, to the co-dehydrogenase.
  2. The coenzyme passes it to the oxaloacetic acid*.
  3. The malic acid then passes it on again to a co-enzyme,
  4. which passes the hydrogen to the yellow enzyme.
  5. The yellow enzyme passes the hydrogen to the fumarate.
  6. The succinate so produced is then oxidized by cytochrome,
  7. the cytochrome by respiratory enzyme,
  8. the respiratory enzyme by oxygen.

So the reaction 2H + O – H2O, which seems such a simple one,

  • breaks down into a long series of separate reactions.

With each new step, with each transfer between substances,

  • the hydrogen loses some of its energy,
  • finally combining with oxygen in its lowest-energy compound.

So each hydrogen atom is gradually oxidized in a long series of reactions, and

  • its energy released in stages.

This oxidation of hydrogen in stages seems to be one of the basic principles of biological oxidation. The reason for it is probably mainly that

  • the cell would not be able to harness and transfer to other processes
  • the large amount of energy which would be released by direct oxidation.

The cell needs small change if it is to be able to

  • pay for its functions without losing too much in the process.

So it oxidizes the H-atom by stages, converting the large banknote into small change.

About half of all plants – contain a polyphenol, generally a pyrocatechol derivative, together with an enzyme, polyphenoloxidase, which oxidizes polyphenol with the help of oxygen. The current interpretation of the mode of action of this oxidase was a confused one. I succeeded in showing that the situation was simply this, that

the oxidase oxidizes the polyphenol to quinone with oxygen.

  • In the intact plant the quinone is reduced back again
  • with hydrogen made available from the foodstuff.

Phenol therefore acts as a hydrogen-carrier between oxygen and the H-donor, and we are here again faced with a probably still imperfectly understood system for

  • the stepwise combustion of hydrogen.


Vitamin C

If benzidine is added to a peroxide in the presence of peroxidase, a deep-blue color appears immediately, which is caused by the oxidation of the benzidine. This reaction does not occur without peroxidase. I simply used some juice which had been squeezed from these plants instead of a purified peroxidase, and added benzidine and peroxide, and the blue pigment appeared, after a small delay of about a second. Analysis of this delay showed that it was due to the presence of a powerful reducing substance, which reduced the oxidized benzidine again, until it had itself been used up. Thanks to the invitation from F. G. Hopkins and the help of the Rockefeller Foundation, I was able ten years ago to transfer my workshop to Cambridge, where for the first time I was able to pay more serious attention to chemistry. Soon I succeeded in isolating the substance in question from adrenals and various plants, and in showing that it corresponded to the formula C6H8O6 and was related to the carbohydrates. This last circumstance induced me to apply to Prof. W. N. Haworth, who immediately recognized the chemical interest of the substance and asked me for a larger quantity to permit analysis of its structure.

The Mayo Foundation and Prof. Kendall came to my help on a large scale, and made it possible for me to work, regardless of expense, on the material from large American slaughter-houses. The result of a year’s

work-was 25 g of a crystalline substance, which was given the name “hexuronic acid”. I shared this amount of the substance with Prof. Haworth. He undertook to investigate the exact structural formula of the substance. I used the other half of my preparation to gain a deeper understanding of the substance’s function. The substance could not replace the adrenals, but caused the disappearance of pigmentation in patients with Addison’s disease.

In 1930 I settled down in my own country at the University of Szeged. I also received a first-rate young American collaborator, J. L. Svirbely, who had experience in vitamin research, but besides this experience brought only the conviction that my hexuronic acid was not identical with vitamin C. In the autumn of 1931 our first experiments were completed, and showed unmistakably that hexuronic acid was power- fully anti-scorbutic, and that the anti-scorbutic acitvity of plant juices corresponded to their hexuronic acid content. We did not publish our results till the following year after repeating our experiments. At this time Tillmans was already directing attention to the connection between the reducing strength and the vitamin activity of plant juices. At the same time King and Waugh also reported crystals obtained from lemon juice, which were active anti-scorbutically and resembled our hexuronic acid.

My town, Szeged, is the centre of the Hungarian paprika industry. Since this fruit travels badly, I had not had the chance of trying it earlier. The sight of this healthy fruit inspired me one evening with a last hope, and that same night investigation revealed that this fruit represented an unbelievably rich source of hexuronic acid, which, with Haworth, I re-baptized ascorbic acid. I also had the privilege of providing my two prize-winning colleagues P. Karrer and W. N. Haworth with abundant material, and making its structural analysis possible for them. I myself produced with Varga the mono-acetone derivative of ascorbic acid, which forms magnificent crystals; from which, after repeated dissolving and recrystallization, ascorbic acid can be separated again with undiminished activity. This was the first proof that ascorbic acid was identical with vitamin C.

Returning to the processes of oxidation, I now tried to analyse further the system of respiration in plants, in which ascorbic acid and peroxidase played an important part. I had already found in Rochester that the peroxidase plants contain an enzyme which reversibly oxidizes ascorbic acid with two valencies in the presence of oxygen. Further analysis showed that here again a system of respiration was in question, in which hydrogen was oxidized by stages. I would like, in the interests of brevity, to summarize the end result of these experiments, which I carried out with St. Huszák. Ascorbic acid oxidase oxidizes the acid with oxygen to reversible dehydroascorbic acid, whereby the oxygen unites with the two labile H-atoms from the acid to form hydrogen peroxide. This peroxide reacts with peroxidase and oxidizes a second molecule of ascorbic acid. Both these molecules of dehydro-ascorbic acid again take up hydrogen from the foodstuff, possibly by means of SH-groups. But peroxidase does not oxidize ascorbic acid directly. Another substance is interposed between the two, which belongs to the large group of yellow, water-soluble phenol-benzol-r-pyran plant dyes (flavone, flavonol, flavanone). Here the peroxidase oxidizes the phenol group to the quinone, which then oxidizes the ascorbic acid directly, taking up both its H-atoms.

At the time that I had just detected the rich vitamin content of the paprika, I was asked by a colleague of mine for pure vitamin C. This colleague himself suffered from a serious haemorrhagic diathesis. Since I still did not have enough of this crystalline substance at my disposal then, I sent him paprikas. My colleague was cured. But later we tried in vain to obtain the same therapeutic effect with pure vitamin C. Guided by my earlier studies into the peroxidase system, I investigated with my friend St. Rusznyák and his collaborators Armentano and Bentsáth the effect of the other link in the chain, the flavones. Certain members of this group of substances, the flavanone hesperidin (Fig. 5) and the formerly unknown eriodictyolglycoside, a mixture of which we had isolated from lemons and named citrin, now had the same therapeutic effect as paprika itself.

H U G O T H E O R E L L          Nobel Lecture, December 12, 1955

The nature and mode of action of oxidation enzymes


Practically all chemical reactions in living nature are started and directed in their course by enzymes. This being the case, Man has of course since time immemorial seen examples of what we now call enzymatic reactions, e.g. fermentation and decay. It would thus be possible to trace the history of enzymes back to the ancient Greeks, or still further for that matter. But it would be rather pointless, since to observe a phenomenon is not the same thing as to explain it. It is more correct to say that our knowledge of enzymes is essentially a product of twentieth-century research.

Jöns Jacob Berzelius, wrote in his yearbook in 1835: “…The catalytic force appears actually to consist thought herein that through their mere presence, and not through their affinity, bodies are able to arouse affinities which at this temperature are slumbering…”  Enzymes are the catalyzers of the biological world, and Berzelius’ description of catalytic force is surprisingly far-sighted…  if one could once understand the mechanism it would doubtless prove that the forces of ordinary chemistry would suffice to explain also these as yet mysterious reactions.

The year 1926 was a memorable one. The German chemist Richard Willstitter gave a lecture then in Deutsche Chemische Gesellschaft, in which he summarized the experiences gained in his attempts over many years to produce pure enzymes. Willstätter drew the conclusion that the enzymes did not belong to any known class of chemical substances, and that the effects of the enzymes derived from a new natural force, thus taking the view that 90 years earlier Berzelius thought to be improbable. That same year, through an irony of fate, the American researcher J. B. Sumner published a work in which he claimed to have crystallized in pure form an enzyme, urease. In the ensuing years J. H. Northrop and his collaborators crystallized out a further three enzyme preparations, pepsin, trypsin, and chymotrypsin, like urease, hydrolytic enzymes that split linkages by introducing water. If these discoveries had been undisputed from the outset it would probably not have been 20 years before Sumner, together with Northrop and Stanley, received a Nobel Prize.

When in 1933 I went on a Rockefeller fellowship to Otto Warburg’s institute in Berlin, Warburg and Christian had in the previous year produced a yellow-coloured preparation of an oxidation enzyme from yeast. The yellow colour was of particular interest: it faded away on reduction and returned on oxidation with e.g. oxygen, so that it was evident that the yellow pigment had to do with the actual enzymatic process of oxido-reduction. It was possible to free the yellow pigment from the high-molecular carrier substance, whose nature was still unknown, for example by treatment with acid methyl alcohol, whereupon the enzyme effect disappeared. Through simultaneous works by Warburg in Berlin, Kuhn in Heidelberg and Karrer in Zurich the constitution of the yellow pigment (lactoflavin, later riboflavin or vitamin B,) was determined. It was here for the first time possible to localize the enzymatic effect to a definite atomic constellation: hydrogen freed from the substrate (hexose monophosphate) is, with the aid of a special enzyme system (TPN-Zwischenferment) whose nature was elucidated somewhat later, placed on the nitrogen atoms of the flavin (1) and (10), giving rise to the colourless leucoflavin. This is reoxidized by oxygen, hydrogen peroxide being formed, and may afterwards be reduced again, and so forth. This cyclic process then continues until the entire amount of substrate has been deprived of two hydrogen atoms and been transformed into phosphogluconic acid; and a corresponding amount of hydrogen peroxide has been formed. At the end of the process the yellow enzyme is still there in unchanged form, and has thus apparently, as Berzelius expressed himself, aroused a chemical affinity through its mere presence.

The polysaccharides, which constituted 80-90% of the entire weight, were completely removed, together with some inactive colourless proteins. After fractionated precipitations with ammonium sulphate I produced a crystalline preparation which on ultracentrifuging and electrophoresis appeared homogeneous. The enzyme was a protein with the molecular weight 75,000 and strongly yellow-colored by the flavin part. The result of the Flavin analysis was 1 mol flavin per 1 mol protein. With dialysis against diluted hydrochloric acid at low temperature the yellow pigment was separated from the protein, which then became colorless. In the enzyme test the flavin part and the protein separately were inactive, but if the flavin part and the protein were mixed at approximately neutral reaction the enzyme effect returned, and the original effect came back when one mixed them in the molecular proportions 1 : 1. That in this connection a combination between the pigment and the protein came about was obvious, moreover, for other reasons: the green-yellow colour of the flavin part changed to pure yellow,and its strong. yellow fluorescence disappeared with linking to the protein.

In my electrophoretic experiments lactoflavin behaved as a neutral body, while the pigment part separated from the yellow enzyme moved rapidly towards the anode and was thus an acid. An analysis for phosphorus showed 1 P per mol flavin, and when after a time (1934) I succeeded in isolating the natural pigment component this proved to be a lactoflavin phosphoric acid ester, thus a kind of nucleotide, and it was obvious that the phosphoric acid served to link the pigment part to the protein. I will now show some simple experiments with the yellow enzyme, its colored part, which we now generally refer to as FMN (flavin mononucleotide), and the colorless enzyme protein.

  • The ferment-solution is pure yellow, the FMN-solution green-yellow,owing to the 1st that the light-absorption band in the blue of the free FMN is displaced somewhat in the long-wave direction on being linked with the protein component. A reducing agent (Na2S2O4) is now added to the one cuvette, it is indifferent which. The colour disappears in consequence of the formation of leucoflavin. Oxygen-gas is bubbled through the solution: the colour comes back as soon as the excess of reducing agent has been consumed. The experiment demonstrates the reaction cycle of the yellow enzyme: reduction through hydrogen from the substrate side, reoxidation with oxygen-gas.
  • A flask containing FMN-solution so diluted that its yellow color is not descernible to the eye is placed on a lamp giving long-wave ultraviolet light. The solution gives a strong, yellow fluorescence which disappears on reduction and returns on bubbling with oxygen-gas.
  • Two flasks are placed on the fluorescence lamp. The one contains a diluted solution of the free protein in phosphate buffer (pH 7), the other phosphate buffer alone. An equal amount of FMN-solution is dripped into each flask. In the flask with protein the fluorescence is at once extinguished,

but in the flask with buffer-solution alone it remains. The experiment demonstrates the resynthesis of  yellow enzyme, and since the fluorescence is extinguished by the protein, one may draw the conclusion that some group in the protein is in this connection linked to the imino-group NH(3) of the flavin, which according to Kuhn must be free for the fluorescence to appear.

The significance of these investigations on the yellow enzyme may be summarized

as follows.

  1. The reversible splitting of the yellow enzyme to apo-enzyme + coenzyme in the simple molecular relation 1 : 1 proved that we had here to do with a pure enzyme; the experiments would have been incomprehensible if the enzyme itself had been only an impurity.
  2. This enzyme was thus demonstrably a protein. In the sequel all the enzymes which have been isolated have proved to be proteins.
  3. The first coenzyme, FMN, was isolated and found to be a vitamin phosphoric acid ester. This has since proved to be something occurring widely in nature: the vitamins nicotinic acid amide, thiamine and pyridoxine form in an analogous way nucleotide-like coenzymes, which like the nucleic acids

themselves combine reversibly with proteins.

During the past 20 years a large number of flavoproteins with various enzyme effects have been produced. Instead of FMN many of them contain a dinucleotide, FAD, which consists of FMN + adenylic acid.

We constructed a very sensitive apparatus to record changes in the intensity of the fluorescence, and were thus able to follow the rapidity with which the fluorescence diminishes when FMN and protein are combined, or increases when they are split. Under suitable conditions the speed of combination is very high. Thanks to the great sensitivity of the fluorescent method my Norwegian collaborator Agnar Nygaard and I were able to make accurate determinations of the speed-constant simply by working in extremely diluted solutions, where the speed of combination is low because an FMN molecule so seldom happens to collide with a protein-molecule. We then varied the degree of acidity, ionic milieu and temperature, and we treated the protein with a large number of different reagents which affect in a known way different groups in proteins. In this way we succeeded with a rather high degree of certainty in ascertaining that phosphoric acid in FMN is linked to primary amino-groups in the protein, and the imino-group (3) in FMN to the phenolic hydroxyl group in a tyrosine residue, whereby the fluorescence is extinguished.

We still do not quite understand how through its linkage to the coenzyme the enzyme-protein “activates” the latter to a rapid absorption and giving off of hydrogen. But something we do know. The so-called oxido-reduction potential of the enzyme is in any case of great importance, and it is determined by a simple relation to the dissociation constants for the oxidized and for the reduced coenzyme-enzyme complex. The dissociation constants are in their turn functions of the velocity constants for the combination between coenzyme and enzyme and for the reverse process, and these velocity constants we have been able to determine both in the yellow ferment and in a number of enzyme systems. Without going into any details I may mention that the linkage of coenzyme to enzyme was found to have surprisingly big effects upon the potential of the former.

Alcohol dehydrogenase


Alcohol dehydrogenases occur in both the animal and the vegetable kingdoms, e.g. in liver, in yeast, and in peas. They are colourless proteins which together with DPN may either oxidize alcohol to aldehyde, as occurs chiefly in the liver, or conversely reduce aldehyde to alcohol, as occurs in yeast.

The yeast enzyme was crystallized by Negelein & Wulf (1936) in Warburg’s institute, the liver enzyme (from horse liver) by Bonnichsen & Wassén at our institute in Stockholm in 1948. These two enzymes have come to play a certain general rôle in biochemistry on account of the fact that it has been possible to investigate their kinetics more accurately than is the case with other enzyme systems. The liver enzyme especially, we have on repeated occasions studied with particular thoroughness, since especially favourable experimental conditions here presented themselves. For all reactions with DPN-system it is possible to follow the reaction DPN+ + 2H =+ DPNH + H+ spectrophotometrically, since DPNH has an absorption-band in the more long-wave ultraviolet region, at 340 rnp, and thousands of such experiments have been performed all over the world. A couple of years ago, moreover, we began to apply our fluorescence method, which is based on the fact that DPNH but not DPN fluoresces, even if considerably more weakly than the flavins. Asregards the liver enzyme there is a further effect, which proved extremely useful for certain spectrophotometrical determinations of reaction speeds; together with Bonnichsen I found in 1950 that the 340 rnp band of the reduced coenzyme was displaced, on combination with liver alcohol dehydrogenase, to 325 rnp, and together with Britton Chance we were thus able with the help of his extremely refined rapid spectrophotometric methods to determine the velocity constant for this very rapid reaction. This reaction belongs to the 3 bost problem involving the enzyme, the coenzyme, and the substrate, and both the coenzyme and the substrate occur in both oxidized and reduced forms.

It is a curious whim of nature that the same coenzyme which in the yeast makes alcohol by attaching hydrogen to aldehyde also occurs in the liver to remove from alcohol the same hydrogen, so that the alcohol becomes aldehyde again, which is then oxidized further
Heme proteins

In 1936 we had obtained cytochrome approximately 80% pure, and in 1939 close to 100%.It is a beautiful red, iron-porphyrin-containing protein which functions as a link in the chain of the cell-respiration enzymes, the iron atom now taking up and now giving off an electron, and the iron thus alternating valency between the 3-valent ferri and the 2-valent ferro stages. It is a very pleasant substance to work with, not merely because it is lovely to look at, but also because it is uncommonly stable and durable. From 100 kg horse heart one can produce 3-4 grams of pure cytochrome c. The molecule weighs about 12,000 and contains one mol iron porphyrin per-mol.

Exp. 4. Two cuvettes each contain a solution of ferricytochrome c. The colour is blood-red. To the one are added some grains of sodium hydrosulphite: the color is changed to violet-red (ferrocytochrome). Oxygen is now bubbled through the ferrocytochrome-solution: no visible change occurs. The ferrocyto-chrome can thus not be oxidized by oxygen. A small amount of cytochrome oxidase is now added: the ferricytochrome color returns.

From this experiment we can draw the conclusion that reduced cytochrome c cannot react with molecular oxygen. In a chain of oxidation enzymes it will thus not be able to be next to the oxygen. The incapacity of cytochrome to react with oxygen was a striking fact that required an explanation. Another peculiarity was the extremely firm linkage between the red heme pigment and the protein part; in contradistinction to the majority of other heme protides, the pigment cannot be split off by the addition of acetone acidified with hydrochloric acid. Further, there was a displacement of the light-absorption bands which indicated that the two unsaturated vinyl groups occurring in ordinary protohemin were saturated in the hematin of

the cytochrome. In 1938 we succeeded in showing that the porphyrin part of the cytochrome was linked to the protein by means of two sulphur bridges from cysteine residues in the protein of the porphyrin in such a way that the vinyl groups were saturated and were converted to α-thioether groups. The firmness of the linkage and the displacement of the spectral bands were herewith explained. This was the first time that it had been possible to show the nature of chemical linkages between a “prosthetic” group (in this case iron porphyrin) and the protein part in an enzyme.

The light-absorption bands of the cytochrome showed that it is a so-called hemochromogen, which means that two as a rule nitrogen-containing groups are linked to the iron, in addition to the four pyrrol-nitrogen atoms in the porphyrin. From magnetic measurements that I made at Linus Pauling’s institute in Pasadena and from amino-acid analyses, titration curves and spectrophotometry together with Å. Åkeson it emerged (1941) that the nitrogen-containing, hemochromogen-forming groups in cytochrome c were histidine residues, or to be more specific, their imidazole groups.   Recently we have got a bit farther. Tuppy & Bodo in Vienna began last year with Sanger’s method to elucidate the amino-acid sequence in the hemin-containing peptide fragment that one obtains with the proteolytic breaking down of cytochrome c, and succeeded in determining the sequence of the amino acids nearest the heme. The experiments were continued and supplemented by Tuppy, Paléus & Ehrenberg at our institute in Stockholm with the following result:

The peptide chain 1-12 (“Val”) = the amino acid valine, “Glu” = glutamine,”Lys” = lysine, and so forth) is by means of two cysteine-S-bridges and a linkage histidine-Fe linked to the heme. When in 1954 Linus Pauling delivered his Nobel Lecture in Stockholm he showed a new kind of models for the study of the steric configuration of peptide chains, which as we know may form helices or “pleated sheets” of various kinds. It struck me then that it would be extremely interesting to study the question as to which of these possibilities might be compatible with the sulphur bridges to the hemin part and with the linkage of nitrogen containing groups to the iron. Pauling was kind enough to make me a present of his peptide-model pieces, which I shall show presently. This is thus the second time they figure in a Lecture.

Anders Ehrenberg and I now made a hemin model on the same scale as the peptide pieces and constructed models of hemin peptides with every conceivable variant of hydrogen bonding. It proved that many variants could be definitely excluded on steric grounds, and others were improbable for other reasons. Of the original, at least 20 alternatives, finally only one remained – a left-twisting a-helix with the cysteine residue no. 4 linked to the porphyrin side-chain in 4-position, and cysteine no. 7 to the side-chain in 2-position. The imidazole residue fitted exactly to linkage with the iron atom. The peptide spiral becomes parallel with the plane of the heme disc.

Through calculations on the basis of the known partial specific volume of the cytochrome we now consider it extremely probable that the heme plate in cytochrome c is surrounded by peptide spirals on all sides in such a way that the heme iron is entirely screened off from contact with oxygen; here is the explanation of our experiment in which we were unable to oxidize reduced cytochrome c with oxygen-gas. The oxygen simply cannot get at the iron atom. There is, on the other hand, a possibility for electrons to pass in and out in the iron atom via the imidazole groups.  It strikes us as interesting that even at this stage the special mode of reacting of the cytochrome is beginning to be understood from what we know of its chemical constitution.

F r i t z  L i p m a n n           Nobel Lecture, December 11, 1953

Development of the acetylation problem: a personal account


In my development, the recognition of facts and the rationalization of these facts into a unified picture, have interplayed continuously. After my apprenticeship with Otto Meyerhof, a first interest on my own became the phenomenon we call the Pasteur effect, this peculiar depression of the wasteful fermentation in the respiring cell. By looking for a chemical explanation of this economy measure on the cellular level, I was prompted into a study of the mechanism of pyruvic acid oxidation, since it is at the pyruvic stage where respiration branches off from fermentation. For this study I chose as a promising system a relatively simple looking pyruvic acid oxidation enzyme in a certain strain of Lactobacillus delbrueckii1. The decision to explore this particular reaction started me on a rather continuous journey into partly virgin territory to meet with some unexpected discoveries, but also to encounter quite a few nagging disappointments

The most important event during this whole period, I now feel, was the accidental observation that in the L. delbrueckii system, pyruvic acid oxidation was completely dependent on the presence of inorganic phosphate. This observation was made in the course of attempts to replace oxygen by methylene blue. To measure the methylene blue reduction manometrically, I had to switch to a bicarbonate buffer instead of the otherwise routinely used phosphate. In bicarbonate, to my surprise, as shown in Fig. 1, pyruvate oxidation was very slow, but the addition of a little phosphate caused a  remarkable increase in rate. The next figure, Fig. 2, shows the phosphate effect more drastically, using a preparation from which all phosphate was removed by washing with acetate buffer. Then it appeared that the reaction was really fully dependent on phosphate. In spite of such a phosphate dependence, the phosphate balance measured by the ordinary Fiske-Subbarow procedure did not at first indicate any phosphorylative step. Nevertheless, the suspicion remained that phosphate in some manner was entering into the reaction and that a phosphorylated intermediary was formed. As a first approximation, a coupling of this pyruvate

oxidation with adenylic acid phosphorylation was attempted. And, indeed, addition of adenylic acid to the pyruvic oxidation system brought out a net disappearance of inorganic phosphate, accounted for as adenosine triphosphate (Table 11). In parallel with the then just developing fermentation now concluded that the missing link in the reaction chain was acetyl phosphate. In partial confirmation it was shown that a crude preparation of acetyl phosphate, synthesized by the old method of Kämmerer and Carius2

would transfer phosphate to adenylic acid (Table 2). However, it still took quite some time from then on to identify acetyl phosphate definitely as the initial product of the pyruvic oxidation in this system3,4.

At the time when these observations were made, about a dozen years ago, there was, to say the least, a tendency to believe that phosphorylation was rather specifically coupled with the glycolytic reaction. Here, however, we had found a coupling of phosphorylation with a respiratory system. This observation immediately suggested a rather sweeping biochemical significance, of transformations of electron transfer potential, respiratory or fermentative, to phosphate bond energy and therefrom to a wide range of biosynthetic reactions7.

There was a further unusual feature in this pyruvate oxidation system in that the product emerging from the process not only carried an energy-rich phosphoryl radical such as already known, but the acetyl phosphate was even more impressive through its energy-rich acetyl. It rather naturally became a contender for the role of “active” acetate, for the widespread existence of which the isotope experience had already furnished extensive evidence. I became, therefore, quite attracted by the possibility that acetyl phosphate could serve two rather different purposes, either to transfer its phosphoryl group into the phosphate pool, or to supply its active acetyl for biosynthesis of carbon structures. Thus acetyl phosphate should be able to serve as acetyl donor as well as phosphoryl donor, transferring, as shown in Fig. 3, on either side of the oxygen center, such as indicated by Bentley’s early experiments on cleavage7a of acetyl phosphate in H2 18O.

These two novel aspects of the energy problem, namely

(1) the emergence of an energy-rich phosphate bond from a purely respiratory reaction; and

(2) the presumed derivation of a metabolic building-block through this same there towards a general concept of transfer of activated groupings by carrier as the fundamental reaction in biosynthesis8,9.

Although in the related manner the appearance of acetyl phosphate as a metabolic intermediary first

focussed attention to possible mechanisms for the metabolic elaboration of group activation, it soon turned out that the relationship between acetyl phosphate and acetyl transfer was much more complicated than anticipated. reaction, prompted me to propose

  • not only the generalization of the phosphate bond as a versatile energy distributing system,
  • but also to aim there towards a general concept of transfer of activated groupings by carrier as the fundamental reaction in biosynthesis8,9.

Although in the related manner the appearance of acetyl phosphate as a metabolic intermediary first focussed attention to possible mechanisms for the metabolic elaboration of group activation, it soon turned out that the relationship between acetyl phosphate and acetyl transfer was much more complicated than anticipated.

It appeared that as an energy source the particle bound oxidative phosphorylation of the kind observed first by Herman Kalckar14 could be replaced by ATP, as had first been observed with the acetylation of choline in brain preparations by Nachmansohn and his group15,16. Using ATP and acetate as precursors, it was possible to set up a homogeneous particle-free acetylation system obtained by extraction of acetone pigeon liver. In this extract acetyl phosphate was unable to replace the ATP acetate as acetyl precursor.

In spite of this disappointment with acetyl phosphate, our decision to turn to a study of acetylation started then to be rewarding in another way. During these studies we became aware of the participation of a heat-stable factor which disappeared from our enzyme extracts on aging or dialysis. This cofactor was present in boiled extracts of all organs, as well as in microorganisms and yeast. It could not be replaced by any other known cofactor. Therefore, it was suspected that we were dealing with a new coenzyme. From then on, for a number of years, the isolation and identification of this coenzyme became the prominent task of our laboratory. The problem now increased in volume and I had the very good fortune that a group of exceedingly able people were attracted to the laboratory; first Constance Tuttle, then Nathan O. Kaplan and shortly afterwards, G. David Novelli, and then others.

Early data on the replacement of this heat-stable factor by boiled extracts are shown in the next table (Table 3). The pigeon liver acetylation system proved to be a very convenient assay system for the new coenzyme17 since on aging for 4 hours at room temperature, the cofactor was completely autolyzed.

Fortunately, on the other hand, the enzyme responsible for the decomposition of this factor was quite unstable and faded out during the aging, while the acetylation apoenzymes were unaffected.

The next figure, Fig. 4, shows coenzyme A (CoA) assay curves obtained with acetone pigeon liver extract. Finding pig liver a good source for the coenzyme, we set out to collect a reasonably large quantity of a highly purified preparation and then to concentrate on the chemistry with this material. In this analysis we paid particular attention to the possibility of finding in this obviously novel cofactor one of the vitamins.

The subsequence finding of a B-vitamin in the preparation gave us further confidence that we were dealing here with a key substance. We still felt, however, slightly dissatisfied with the proof for pantothenic acid. Therefore, to liberate the chemically rather unstable pantothenic acid from CoA, we made use of observations on enzymatic cleavage of the coenzyme. Two enzyme preparations, intestinal phosphatase and an enzyme in pigeon liver extract, had caused independent inactivation. It then was found that through combined action of these two enzymes, pantothenic acid was liberated18,19.

The two independent enzymatic cleavages indicated early that in CoA existed two independent sites of attachment to the pantothenic acid molecule. One of these obviously was a phosphate link, linking presumably to one of a hydroxyl group in pantothenic acid. The other moiety attached to pantothenic acid, which, cleaved off by liver enzyme, remained unidentified for a long time. In addition to pantothenic acid, our sample of 40 per cent purity had been found to contain about 2 per cent sulfur by elementary analysis and identified by cyanide-nitroprusside test as a potential SH grouping 20,21. Furthermore, the coenzyme preparation contained large amounts of adenylic acid21.

Units Coenzyme

Fig. 4. Concentration-activity curves for coenzyme A preparations of different purity. The arrow indicates the point of 1 unit on the curve. (o) crude coenzyme, 0.25 unit per mg; (x) purified coenzyme, 130 units per mg.

In the subsequent elaboration of the structure, the indications by enzyme analysis for the two sites of attachment to pantothenic acid have been most helpful. The phosphate link was soon identified as a pyrophosphate bridge22; 5-adenylic acid was identified by Novelli23 as enzymatic split product and by Baddiley 24, through chemical cleavage. At the same time, Novelli made observations which indicated the presence of a third phosphate in addition to the pyrophosphate bridge. These indications were confirmed by analysis of a nearly pure preparation which was obtained by Gregoryas from Streptomyces fradiae in collaboration with the research group at the Upjohn Company26.

It was at this period that we started to pay more and more attention to the sulfur in the coenzyme. As shown in Table 5, our purest preparation contained 4.13 per cent sulfur corresponding to one mole per mole of pantothenate. We also found26 that dephosphorylation of CoA yielded a compound containing pantothenic acid and the sulfur carrying moiety, which we suspected as bound through the carboxyl. Through the work of Snell and his group27, the sulfur-containing moiety proved to be attached to pantothenic acid through a link broken by our liver enzyme. It was identified as thioethanolamine by Snell and his group, linked peptidically to pantothenic acid.

Through analysis and synthesis, Baddiley now identified the point of attachment of the phosphate bridge to pantothenic acid in 4-position24 and Novelli et al.28 completed the structure analysis by enzymatic synthesis of “dephospho-CoA” from pantetheine-4’-phosphates and ATP. Furthermore, the attachment of the third phosphate was identified by Kaplan29 to attach in s-position on the ribose of the 5-adenylic acid (while in triphosphopyridine nucleotide it happens to be in 2-position). Therefore, the structure was now

established, as shown in Fig. 5.

Fig. 5. Structure of coenzyme A


The metabolic function of CoA

Parallel with this slow but steady elaboration of the structure, all the time we explored intensively metabolic mechanisms in the acetylation field. By use of the enzymatic assay, as shown in Tables 6, 7, 8, and 9, CoA was found present in all living cells, animals, plants and microorganisms17. Furthermore,

the finding that all cellular pantothenic acid could be accounted for by CoA17 made it clear that CoA represented the only functional form of this vitamin. The finding of the vitamin furnished great impetus; nevertheless, a temptation to connect the pantothenic acid with the acetyl transfer function has

blinded us for a long time to other possibilities.

The first attempts to further explore the function of CoA were made with pantothenic acid-deficient cells and tissues. A deficiency of pyruvate oxidation in pantothenic acid-deficient Proteus morganii, an early isolated observation by Dorfman30 and Hills31, now fitted rather well into the picture. We soon became quite interested in this effect, taking it as an indication for participation of CoA in citric acid synthesis. A parallel between CoA levels and pyruvate oxidation in Proteus morganii was demonstrated32. Using panto thenic aciddeficient yeast, Novelli et al.33 demonstrated a CoA-dependence of acetate oxidation (Fig. 5a) and Olson and Kaplan34 found with duck liver a striking parallel between CoA content and pyruvic utilization, which is shown in Fig. 6.

But more important information was being gathered on -the enzymatic level. The first example of a generality of function was obtained by comparing the activation of apoenzymes for choline- and sulfonamide-acetylation respectively, using our highly purified preparations9 of CoA. As shown in Fig. 7, similar activation curves obtained for the two respective enzymes. Through these experiments, the heat-stable factor for choline acetylation that had been found by Nachmansohn and Berman35 and by Feldberg and Mann36 was identified with CoA. The next most significant step toward a generalization of CoA function for acetyl transfer was made by demonstrating its functioning in the enzymatic synthesis of acetoacetate. The CoA effect in acetoacetate synthesis was studied by Morris Soodak37, who obtained for this reaction a reactivation curve quite similar to those for enzymatic acetylation, as shown in Fig. 8.

Soon afterwards Stern and Ochoa38 showed a CoA-dependent citrate synthesis with a pigeon liver fraction similar to the one used by Soodak for acetoacetate synthesis. In our laboratory, Novelli et al. confirmed and extended this observation with extracts of Escherichia coli39.

In the course of this work, which more and more clearly defined the acetyl transfer function of CoA, Novelli once more tried acetyl phosphate. To our surprise and satisfaction, it then appeared, as shown in Table 9, that in Escherichia coli extracts in contrast to the animal tissue, acetyl phosphate was more than twice as active as acetyl donor for citrate synthesis than ATP acetate 39. Acetyl phosphate, therefore, functioned as a patent microbial acetyl donor. Acetyl transfer from acetyl phosphate, like that from ATP-acetate, was CoA-dependent, as shown in Table 9. Furthermore, a small amount of “microbial conversion factor”, as we called it first, primed acetyl phosphate for activity with pigeon liver acetylation systems40, as shown in Table 10.

Eventually the microbial conversion factor was identified by Stadtman et al.40 with the transacetylase first encountered by Stadtman and Barker in extracts of Clostridium kluyveri41 and likewise, although not clearly defined as such, in extracts of Escherichia coli and Clostridium butylicum by Lipmann and Tuttle42. The definition of such a function was based on the work of Doudoroff et al.43 on transglucosidation with sucrose phosphorylase. Their imaginative use of isotope exchange for closer definition of enzyme mechanisms has been most influential. Like glucose-I-phosphate with sucrose phosphorylase, acetyl phosphate with these various microbial preparations equilibrates its phosphate rapidly with the inorganic phosphate of the solution. As in Doudoroff et al. experiments, first a covalent substrate enzyme derivative had been proposed 43. However, then Stadtman et al.40, with the new experience of CoA dependent acetyl transfer, could implicate CoA in this equilibration between acetyl- and inorganic phosphate and thus could define the transacetylase as an enzyme equilibrating acetyl between phosphate and CoA:

In the course of these various observations, it became quite clear that there existed in cellular metabolism an acetyl distribution system centering around CoA as the acetyl carrier which was rather similar to the ATP-centered phosphoryl distribution system. The general pattern of group transfer became recognizable, with donor and acceptor enzymes being connected through the CoA —- acetyl CoA shuttle. A clearer definition of the donor-acceptor enzyme scheme was obtained through acetone fractionation of our standard system for acetylation of sulfonamide into two separate enzyme fractions, which were inactive separately but showed the acetylation effect when combined. A fraction, A-40, separating out with 40 per cent acetone, was shown by Chou44 to contain the donor enzyme responsible for the ATP-CoA-acetate reaction, while with more acetone precipitated, the acceptor function, A-60, the acetoarylamine kinase as we propose to call this type of enzyme. The need for a combination of the two for overall acetyl transfer is shown in Fig. 9. This showed that a separate system was responsible for acetyl CoA formation through interaction of ATP, CoA and acetate (cf. below) and that the overall acetylation was a two-step reaction:

These observations crystallized into the definition of a metabolic acetyl transfer territory as pictured in Fig. 10. This picture had developed from the growing understanding of enzymatic interplay involving metabolic generation of acyl CoA and transfer of the active acyl to various acceptor systems. A most important, then still missing link in the picture was supplied through the brilliant work of Feodor Lynen45 who chemically identified acetyl CoA as the thioester of CoA. Therewith the thioester link was introduced as a new energy-rich bond and this discovery added a very novel facet to our understanding of the mechanisms of metabolic energy transformation.

Enzyme Localization In The  Anaerobic Mitochondria Of Ascaris L Umbricoides


Robert S. Rew And Howard J. Saz

From the Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556


Mitochondria from the muscle of the parasitic nematode Ascaris lumbricoides   var. suum function anaerobically in electron transport-associated  phosphorylations under physiological conditions. These helminth organelles have been fractionated into inner and outer membrane, matrix, and intermembrane space fractions. The distributions of enzyme systems were determined and compared with corresponding distributions reported in mammalian mitochondria.  Succinate and pyruvate dehydrogenases as well as NADH  oxidase, Mg++-dependent ATPase, adenylate kinase, citrate synthase, and cytochrome c  reductases  were  determined to be distributed  as  in mammalian mitochondria.  In contrast  with  the  mammalian systems, fumarase and NAD-linked “malic” enzyme were isolated primarily from the intermembrane  space fraction of the worm mitochondria. These enzymes required for the anaerobic  energy-generating system in Ascaris and would be expected to give rise to NADH in the intermembrane space.  The need for and possible mechanism of a proton translocation system to obtain energy generation is suggested.                                Downloaded from







David Keilin’s Respiratory Chain Concept and its Chemiosmotic Consequences

Peter Mitchell              Nobel Lecture, 8 December, 1978

Glynn Research Institute, Bodmin, Cornwall, U. K.
“for his contribution to the understanding of biological energy transfer through the formulation of the chemiosmotic theory”

Peter D. Mitchell (1920-1992) received the Nobel Prize in 1978 for developing the Chemiosmotic Theory to explain ATP synthesis resulting from membrane-associated electron transport [Ubiquinone and the Proton Pump].

Mitchell is the last of the gentleman scientists. He first proposed the chemiosmotic principle in a 1961 Nature article while he was at the University of Edinburgh. Shortly after that, ill health forced him to move to Cornwall where he renovated an old manor house and converted it into a research laboratory. From then on, he and his research colleague, Jennifer Moyle, continued to work on the chemiosmotic theory while being funded by his private research foundation. [Peter Mitchell: Wikipedia]

The Chemiosmotic Theory was controversial in 1978 and it still has not been fully integrated into some biochemistry textbooks in spite of the fact that it is now proven. The main reason for the resistance is that it overthrows much of traditional biochemistry and introduces a new way of thinking. It is a good example of a “paradigm shift” in biology.

Because he was such a private, and eccentric, scientist there are very few photos of Peter Mitchell or his research laboratory at Glynn House . The best description of him is in his biography Wandering in the Gardens of the Mind: Peter Mitchell and the Making of Glynn by John Prebble, and Bruce Weber. A Nature review by E.C. Slater [Metabolic Gardening] gives some of the flavor and mentions some of the controversy.





Many scientists believe that the Chemiosmotic Theory was the second greatest contribution to biology in the 2oth century (after the discovery of the structure of DNA). Mitchell had to overcome many critics including Hans Krebs. The case is strong.

In the 1960s, ATP was known to be the energy currency of life, but the mechanism by which ATP was created in the mitochondria was assumed to be by substrate-level phosphorylation. Mitchell’s chemiosmotic hypothesis was the basis for understanding the actual process of oxidative phosphorylation. At the time, the biochemical mechanism of ATP synthesis by oxidative phosphorylation was unknown.

Mitchell realised that the movement of ions across an electrochemical potential difference could provide the energy needed to produce ATP. His hypothesis was derived from information that was well known in the 1960s. He knew that living cells had a membrane potential; interior negative to the environment. The movement of charged ions across a membrane is thus affected by the electrical forces (the attraction of positive to negative charges). Their movement is also affected by thermodynamic forces, the tendency of substances to diffuse from regions of higher concentration. He went on to show that ATP synthesis was coupled to this electrochemical gradient.[11]

His hypothesis was confirmed by the discovery of ATP synthase, a membrane-bound protein that uses the potential energy of the electrochemical gradient to make ATP.

Growth, development and metabolism are some of the central phenomena in the study of biological organisms. The role of energy is fundamental to such biological processes. The ability to harness energy from a variety of metabolic pathways is a property of all living organisms. Life is dependent on energy transformations; living organisms survive because of exchange of energy within and without.

In a living organism, chemical bonds are broken and made as part of the exchange and transformation of energy. Energy is available for work (such as mechanical work) or for other processes (such as chemical synthesis and anabolic processes in growth), when weak bonds are broken and stronger bonds are made. The production of stronger bonds allows release of usable energy.

One of the major triumphs of bioenergetics is Peter D. Mitchell‘s chemiosmotic theory of how protons in aqueous solution function in the production of ATP in cell organelles such as mitochondria.[5] This work earned Mitchell the 1978 Nobel Prize for Chemistry. Other cellular sources of ATP such as glycolysis were understood first, but such processes for direct coupling of enzyme activity to ATP production are not the major source of useful chemical energy in most cells. Chemiosmotic coupling is the major energy producing process in most cells, being utilized in chloroplasts and several single celled organisms in addition to mitochondria.


In August 1960, Robert K. Crane presented for the first time his discovery of the sodium-glucose cotransport as the mechanism for intestinal glucose absorption.[2] Crane’s discovery of cotransport was the first ever proposal of flux coupling in biology and was the most important event concerning carbohydrate absorption in the 20th century.[3][4]

The free energy (ΔG) gained or lost in a reaction can be calculated: ΔG = ΔH – TΔS
where G = Gibbs free energy, H = enthalpy, T = temperature, and S = entropy.

How inositol pyrophosphates control cellular phosphate homeostasis?

Adolfo Saiardi*

Cell Biology Unit, Medical Research Council Laboratory for Molecular Cell Biology, Department of Cell and Developmental Biology,

University College London, Gower Street, London WC1E 6BT, United Kingdom

Advances in Biological Regulation 52 (2012) 351–359

Phosphorus in his phosphate PO43_ configuration is an essential constituent of all life forms. Phosphate diesters are at the core of nucleic acid structure, while phosphate monoester transmits information under the control of protein kinases and phosphatases. Due to these fundamental roles in biology it is not a surprise that phosphate cellular homeostasis is under tight control.

Inositol pyrophosphates are organic molecules with the highest proportion of phosphate groups, and they are capable of regulating many biological processes, possibly by controlling energetic metabolism and adenosine triphosphate (ATP) production.

Furthermore, inositol pyrophosphates influence inorganic polyphosphates (polyP) synthesis. The polymer polyP is solely constituted by phosphate groups and beside other known functions, it also plays a role in buffering cellular free phosphate [Pi] levels, an event that is ultimately necessary to generate ATP and inositol pyrophosphate.

Two distinct classes of proteins the inositol hexakisphosphates kinases (IP6Ks) and the diphosphoinositol pentakisphosphate kinases (PP-IP5Ks or IP7Ks) are capable of synthesizing inositol pyrophosphates.

IP6Ks utilize ATP as a phosphate donor to phosphorylate IP6 to IP7, generation the isomer 5PP-IP5 (Fig. 1A), and inositol pentakisphosphate I(1,3,4,5,6)P5 to PP-IP4 (Saiardi et al., 1999, 2000; Losito et al., 2009). Furthermore, at least in vitro, IP6Ks generate more complex molecules containing two or more pyrophosphate moieties, or even three-phosphate species (Draskovic et al., 2008; Saiardi et al., 2001). Three IP6K isoforms referred to as IP6K1, 2, 3 exist in mammal; however, there is a single IP6K in the yeast Saccharomyces cerevisiae called Kcs1.

The PP-IP5Ks enzymes, synthesize inositol pyrophosphate from IP6, but not from IP5, (Losito et al., 2009) generating the isomer 1PP-IP5. Kinetic studies performed in vitro suggested that IP7, the 5PP-IP5 isomer generated by IP6Ks, is the primary substrate of this new enzyme, and this finding was confirmed in vivo by analysing PP-IP5K null yeast (vip1D) that accumulate the un-metabolized substrate IP7 (Azevedo et al., 2009; Onnebo and Saiardi, 2009). Thus PP-IP5K is responsible for IP8,

isomer 1,5PP2-IP4 synthesis (Fig. 1A). Two PP-IP5K isoforms referred to as PP-IP5Ka and b exist in mammal while a single PP-IP5K called Vip1 is present in S. cerevisiae.

Inositol pyrophosphates are hydrolysed by the diphosphoinositol-polyphosphate phosphohydrolases (DIPPs) (Safrany et al., 1998). Four mammalian enzymes DIPP1,2,3,4 have been identified, while only one DIPP protein exists in S. cerevisiae called Ddp1. These phosphatases are promiscuous enzymes able to hydrolyse inositol pyrophosphate as well as nucleotide analogues, such as diadenosine hexaphosphate (Ap6A) (Caffrey et al., 2000; Fisher et al., 2002). More recently, it has been shown that DIPPs also degrade polyP (Lonetti et al., 2011). Inositol pyrophosphates control the most disparate biological processes, from telomere length to vesicular trafficking. It is conceivable that all these function can be focused on the fact that inositol pyrophosphates are controlling cellular energy metabolism and consequently, ATP production. We have recently, demonstrated that inositol pyrophosphates control glycolysis and mitochondrial oxidative phosphorylation by both inhibiting the glycolytic flux and increasing mitochondrial activity (Szijgyarto et al., 2011).

Another important molecule to briefly introduce is polyP (Fig. 1B). The interested reader is encouraged to read the following comprehensive reviews (Kornberg et al., 1999; Rao et al., 2009). The polyP polymer likely represents a phosphate buffer that is synthesized and degraded in function of the phosphate needs of the cells. Furthermore, it also functions as a chelator of metal ions, thereby regulating cellular cation homeostasis. However, polyP also possesses more classical signalling roles.

In bacteria for example, it influences pathogenicity (Brown and Kornberg, 2008) and in mammalian cells it has been proposed to regulate fibrinolysis and platelet aggregation (Caen and Wu, 2010). In prokaryotes, polyP synthesis is carried out by a family of conserved polyP kinases (PPKs), whereas degradation is mediated by several polyP phosphatases (Rao et al., 2009). In higher eukaryotes polyP synthesis remains poorly characterized.

In humans alteration of phosphate metabolism is implicated in several pathological states. Higher serum phosphate leads to vascular calcification and cardiovascular complications. Although only very small amount of phosphate circulates in the serum, its concentration is tightly regulated and it is independent from dietary phosphorus intake (de Boer et al., 2009). Therefore, it is not surprising that intense research efforts are aimed to elucidate phosphate uptake and metabolism. IP6K2 was initially cloned while searching for a novel mammalian intestinal phosphate transporter that the group of Murer identified as PiUS (Phosphate inorganic Uptake Stimulator) (Norbis et al., 1997). Once transfected into Xenopus oocytes, PiUS stimulated the cellular uptake of radioactive phosphate.

Subsequently, two groups discovered that PiUS was capable of converting IP6 to IP7 and rename it to IP6K2 (Saiardi et al., 1999; Schell et al., 1999). The ability of inositol pyrophosphate to control the uptake of phosphate is an evolutionary conserved feature; in fact, kcs1D yeast with undetectable level of IP7 exhibits a reduced uptake of phosphate from the culture medium (Saiardi et al., 2004).

In mammals, regulation of phosphate homeostasis is not restricted to IP6K2, all three mammalian IP6Ks are likely to play a role. A genome-wide study aimed at identifying genetic variations associated with changes of serum phosphorus concentration identified IP6K3 (Kestenbaum et al., 2010). This human genetic study identified two independent single nucleotide polymorphisms (SNP) at locus 6p21.31, which are localised within the first intron of the IP6K3 gene. Interestingly, this study that analysed more than 16,000 humans identified SNP variant in only seven genes. Three of which, the sodium phosphate cotransporter type IIa, the calcium sensing receptor and the fibroblast growth factor 23, are well known regulators of phosphate homeostasis. These evidences support a role for IP6K3 in controlling serum phosphate levels in humans (Kestenbaum et al., 2010).


The hypothesis


Although, inositol pyrophosphate may have acquired unique organism-specific functions, the conserved ability of this class of molecules to regulate phosphate metabolism suggests an evolutionary ancient role. In this last paragraph, I will formulate few hypotheses that I hope will stimulate further research aimed at elucidating the biological link between phosphate, inositol pyrophosphates and polyP.

Inositol pyrophosphates regulate the entry of phosphate into the cells (Norbis et al., 1997), suggesting that they could affect phosphate uptake either directly (by stimulating a transporter, for example) or a indirectly by helping ‘fixing’ free phosphates in organic molecules. The cytosolic concentration of free phosphate [Pi] cannot fluctuate widely. Therefore, cellular entry of phosphates and its utilization may well be coupled. For example, the synthesis of polyP may be linked to phosphate entry in the cell. Inositol pyrophosphate control of energy metabolism (Szijgyarto et al., 2011) affects not only ATP levels but it can also alter the entire cellular balance of adenine nucleotides. Given that phosphate transfer reactions mainly use ATP as a vehicle for the phosphate groups, inositol pyrophosphate could affect phosphate metabolism by regulating the adenylate cellular pool. Moreover, it is tempting to speculate the existence of a feedback mechanism that coordinates the metabolic balance between ATP, phosphate and inositol pyrophosphates.

Inositol pyrophosphates could either contribute to the regulation of polyP synthesis, play a role in polyP degradation, or both. The yeast polyP polymerase has been identified with the subunit four (Vtc4) of the vacuolar membrane transporter chaperone (VTC) complex (Hothorn et al., 2009). Interestingly, pyrophosphates (Pi–Pi) dramatically accelerate the polyP polymerase reaction. It would therefore be interesting to determine whether the pyrophosphate moiety of IP7 can stimulate polyP vacuolar synthesis in a similar fashion. Similarly, it would be interesting to analyse the effect of inositol pyrophosphates on controlling the activity of the actin-like DdIPK2 enzyme. It should be noted however, that the existence even in yeast or Dictyostelium of other enzymes able to synthesize different polyP pools cannot be excluded. Thus, we will be able to validate and fully appreciate the role played by inositol pyrophosphates on polyP synthesis only after the identification of higher eukaryotes polyp synthesizing peptide/s.

The most abundant form of organic phosphate on earth is IP6, or phytic acid, a molecule that is highly abundant in plant seeds from which was originally characterised. In plant seeds, IP6 represents a phosphate storage molecule that it is hydrolysed during germination, releasing phosphates and cations. It will be an astonishing twist of event if inositol pyrophosphates were controlling the levels of their own precursor IP6 (Raboy, 2003), although due to the evolutionary conserved ability of inositol pyrophosphate to control phosphate homeostasis we should not be entirely surprised.

Although it is not yet clear how inositol pyrophosphates regulate cellular metabolism, understanding how inositol pyrophosphates influence phosphates homeostasis will help to clarify this important link.

Auesukaree C, Tochio H, Shirakawa M, Kaneko Y, Harashima S. Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae. J Biol Chem 2005;280:25127–33.

Azevedo C, Burton A, Ruiz-Mateos E, Marsh M, Saiardi A. Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release. Proc Natl Acad Sci U S A 2009;106:21161–6.

Bennett M, Onnebo SM, Azevedo C, Saiardi A. Inositol pyrophosphates: metabolism and signaling. Cell Mol Life Sci 2006;63:552–64.

Boer VM, Crutchfield CA, Bradley PH, Botstein D, Rabinowitz JD. Growth-limiting intracellular metabolites in yeast growing under diverse nutrient limitations. Mol Biol Cell 2010;21:198–211.

Brown MR, Kornberg A. The long and short of it – polyphosphate, PPK and bacterial survival. Trends Biochem Sci 2008;33:284–90.

Burton A, Hu X, Saiardi A. Are inositol pyrophosphates signalling molecules? J Cell Physiol 2009;220:8–15.

Caen J, Wu Q. Hageman factor, platelets and polyphosphates: early history and recent connection. J Thromb Haemost 2010;8:1670–4.

Caffrey JJ, Safrany ST, Yang X, Shears SB. Discovery of molecular and catalytic diversity among human diphosphoinositol polyphosphate phosphohydrolases. An expanding Nudt family. J Biol Chem 2000;275:12730–6.

A Mitochondrial RNAi Screen Defines Cellular Bioenergetic Determinants and Identifies an Adenylate Kinase as a Key Regulator of ATP Levels

Nathan J. Lanning,1 Brendan D. Looyenga,1,2 Audra L. Kauffman,1 Natalie M. Niemi,1 Jessica Sudderth,3

Ralph J. DeBerardinis,3 and Jeffrey P. MacKeigan1,*

Cell Reports

Altered cellular bioenergetics and mitochondrial function are major features of several diseases, including cancer, diabetes, and neurodegenerative disorders. Given this important link to human health, we sought to define proteins within mitochondria that are critical for maintaining homeostatic ATP levels.

We screened an RNAi library targeting >1,000 nuclear-encoded genes whose protein products localize to the mitochondria in multiple metabolic conditions in order to examine their effects on cellular ATP levels. We identified a mechanism by which electron transport chain (ETC) perturbation under glycolytic conditions increased ATP production through enhanced glycolytic flux, thereby highlighting the cellular potential for metabolic plasticity.

Additionally, we identified a mitochondrial adenylate kinase (AK4) that regulates cellular ATP levels and AMPK signaling and whose expression significantly correlates with glioma patient survival. This study maps the bioenergetic landscape of >1,000 mitochondrial proteins in the context of varied metabolic substrates and begins to link key metabolic genes with clinical outcome.

Comments to be further addressed by JES Roselino

I will add some observations or at least one single observation.
Just at the beginning, when phosphorylation of proteins is presented, I assume you must mention that some proteins are activated by phosphorylation. This is fundamental in order to present self –organization reflex upon fast regulatory mechanisms. Even from an historical point of view. The first observation arrived from a sample due to be studied on the following day of glycogen synthetase. It was unintended left overnight out of the refrigerator. The result was it has changed from active form of the previous day to a non-active form. The story could have being finished here, if the researcher did not decide to spent this day increasing substrate levels (it could be a simple case of denaturation of proteins that changes its conformation despite the same order of amino acids). He kept on trying and found restoration of maximal activity. This assay was repeated with glycogen phosphorylase and the result was the opposite it increases its activity. This lead to the discovery of cAMP activated protein kinase and the assembly of a very complex system in the glycogen granule that is not a simple carbohydrate polymer. Instead it has several proteins assembled and preserves the capacity to receive from a single event (rise in cAMP) two opposing signals with maximal efficiency, stops glycogen synthesis, as long as levels of glucose 6 phosphate are low and increases glycogen phosphorylation as long as AMP levels are high).
I did everything I was able to do by the end of 1970 in order to repeat this assays with PK I, PKII and PKIII of M. Rouxii and Sutherland route to cAMP failed in this case. I ask Leloir to suggest to my chief (SP) the idea of AA, AB, BB subunits as was observed in lactic dehydrogenase (tetramer) indicating this as his idea. The reason was my “chief”(SP) more than once, have said it to me: “Leave these great ideas for the Houssay, Leloir etc…We must do our career with small things.” However, as she also have a faulty ability for recollection she also uses to arrive some time later, with the very same idea but in that case, as her idea.
Leloir, said to me: I will not offer your interpretation to her as mine. I think it is not phosphorylation, however I think it is glycosylation that explains the changes in the isoenzymes with the same molecular weight preserved. This dialogue explains why during “What is life” reading with him he asked me if from biochemist in exile, to biochemist I talked everything to him. Since I have considered that Schrödinger did not have confronted Darlington & Haldane for being in exile. Also, may explain why Leloir could have answered a bad telephone call from P. Boyer, Editor of The Enzymes in a way that suggest the the pattern could be of covalent changes over a protein. Our FEBS and Eur J. Biochemistry papers on pyruvate kinase of M. Rouxii is wrongly quoted in this way on his review about pyruvate kinase of that year(1971).

Another aspect I think you must call attention, in my opinion, is the following, show in detail with different colors what carbons belongs to CoA a huge molecule, in comparison with the single two carbons of acetate that will produce the enormous jump in energy yield in comparison with anaerobic glycolysis. The idea is how much must have being spent in DNA sequences to build that molecule in order to use only two atoms of carbon. Very limited aspects of biology could be explained in this way. In case we follow an alternative way of thinking, it becomes clearer that proteins were made more stable by interaction with other molecules (great and small). Afterwards, it rather easy to understand how the stability of protein-RNA complexes where transmitted to RNA (vibrational +solvational reactivity stability pair of conformational energy). Latter, millions of years, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.


JES Roselino

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Selected References to Signaling and Metabolic Pathways in

Curator: Larry H. Bernstein, MD, FCAP


This is an added selection of articles in Leaders in Pharmaceutical Intelligence after the third portion of the discussion in a series of articles that began with signaling and signaling pathways. There are fine features on the functioning of enzymes and proteins, on sequential changes in a chain reaction, and on conformational changes that we shall return to.  These are critical to developing a more complete understanding of life processes.  I have indicated that many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different.

  1. Signaling and signaling pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence
  4. Lipid metabolism
  5. Protein synthesis and degradation
  6. Subcellular structure
  7. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Selected References to Signaling and Metabolic Pathwayspublished in this Open Access Online Scientific Journal, include the following:

Update on mitochondrial function, respiration, and associated disorders

Curator and writer: Larry H. Benstein, MD, FCAP

A Synthesis of the Beauty and Complexity of How We View Cancer

Cancer Volume One – Summary

A Synthesis of the Beauty and Complexity of How We View Cancer

Author: Larry H. Bernstein, MD, FCAP

Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H Bernstein, MD, FCAP

 The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Author and Curator: Larry H Bernstein, MD, FCAP, 
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
And Curator: Aviva Lev-Ari, PhD, RN

Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

Author and Curator: Larry H. Bernstein, MD, FCAP
Curator:  Stephen J. Williams, PhD
and Curator: Aviva Lev-Ari, PhD, RN

Mitochondrial Metabolism and Cardiac Function

Curator: Larry H Bernstein, MD, FACP

Mitochondrial Dysfunction and Cardiac Disorders

Curator: Larry H Bernstein, MD, FACP

Reversal of Cardiac mitochondrial dysfunction

Curator: Larry H Bernstein, MD, FACP

Advanced Topics in Sepsis and the Cardiovascular System  at its End Stage

Author: Larry H Bernstein, MD, FCAP

Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Curator: Larry H Bernstein, MD, FACP

Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III

Curator: Larry H Bernstein, MD, FCAP


Nitric Oxide, Platelets, Endothelium and Hemostasis (Coagulation Part II)

Curator: Larry H. Bernstein, MD, FCAP

Mitochondrial Damage and Repair under Oxidative Stress

Curator: Larry H Bernstein, MD, FCAP

Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation

Reporter and Curator: Larry H Bernstein, MD, FACP


Nitric Oxide has a Ubiquitous Role in the Regulation of Glycolysis – with a Concomitant Influence on Mitochondrial Function

Reporter, Editor, and Topic Co-Leader: Larry H. Bernstein, MD, FCAP

Mitochondria and Cancer: An overview of mechanisms

Author and Curator: Ritu Saxena, Ph.D.

Mitochondria: More than just the “powerhouse of the cell”

Author and Curator: Ritu Saxena, Ph.D.

Overview of Posttranslational Modification (PTM)

Curator: Larry H. Bernstein, MD, FCAP

Ubiquitin Pathway Involved in Neurodegenerative Diseases

Author and curator: Larry H Bernstein, MD,  FCAP

Is the Warburg Effect the Cause or the Effect of Cancer: A 21st Century View?

Author: Larry H. Bernstein, MD, FCAP

New Insights on Nitric Oxide donors – Part IV

Curator and Author: Larry H. Bernstein, MD, FCAP

Perspectives on Nitric Oxide in Disease Mechanisms [Kindle Edition]

Margaret Baker PhD (Author), Tilda Barliya PhD (Author), Anamika Sarkar PhD (Author), Ritu Saxena PhD (Author), Stephen J. Williams PhD (Author), Larry Bernstein MD FCAP (Editor), Aviva Lev-Ari PhD RN (Editor), Aviral Vatsa PhD (Editor)



Nitric oxide and its role in vascular biology

Signal transmission by a gas that is produced by one cell, penetrates through membranes and regulates the function of another cell represents an entirely new principle for signaling in biological systems.   All compounds that inhibit endothelium-derived relaxation-factor (EDRF) have one property in common, redox activity, which accounts for their inhibitory action on EDRF. One exception is hemoglobin, which inactivates EDRF by binding to it. Furchgott, Ignarro and Murad received the Nobel Prize in Physiology and Medicine for discovery of EDRF in 1998 and demonstrating that it might be nitric oxide (NO) based on a study of the transient relaxations of endothelium-denuded rings of rabbit aorta.  These investigators working independently demonstrated that NO is indeed produced by mammalian cells and that NO has specific biological roles in the human body. These studies highlighted the role of NO in cardiovascular, nervous and immune systems. In cardiovascular system NO was shown to cause relaxation of vascular smooth muscle cells causing vasodilatation, in nervous system NO acts as a signaling molecule and in immune system it is used against pathogens by the phagocytosis cells. These pioneering studies opened the path of investigation of role of NO in biology.

NO modulates vascular tone, fibrinolysis, blood pressure and proliferation of vascular smooth muscles. In cardiovascular system disruption of NO pathways or alterations in NO production can result in preponderance to hypertension, hypercholesterolemia, diabetes mellitus, atherosclerosis and thrombosis. The three enzyme isoforms of NO synthase family are responsible for generating NO in different tissues under various circumstances.

Reduction in NO production is implicated as one of the initial factors in initiating endothelial dysfunction. This reduction could be due to

  • reduction in eNOS production
  • reduction in eNOS enzymatic activity
  • reduced bioavailability of NO

Nitric oxide is one of the smallest molecules involved in physiological functions in the body. It is seeks formation of chemical bonds with its targets.  Nitric oxide can exert its effects principally by two ways:

  • Direct
  • Indirect

Direct actions, as the name suggests, result from direct chemical interaction of NO with its targets e.g. with metal complexes, radical species. These actions occur at relatively low NO concentrations (<200 nM)

Indirect actions result from the effects of reactive nitrogen species (RNS) such as NO2 and N2O3. These reactive species are formed by the interaction of NO with superoxide or molecular oxygen. RNS are generally formed at relatively high NO concentrations (>400 nM)

Although it can be tempting for scientists to believe that RNS will always have deleterious effects and NO will have anabolic effects, this is not entirely true as certain RNS mediated actions mediate important signalling steps e.g. thiol oxidation and nitrosation of proteins mediate cell proliferation and survival, and apoptosis respectively.

  • Cells subjected to NO concentration between 10-30 nM were associated with cGMP dependent phosphorylation of ERK
  • Cells subjected to NO concentration between 30-60 nM were associated with Akt phosphorylation
  • Concentration nearing 100 nM resulted in stabilisation of hypoxia inducible factor-1
  • At nearly 400 nM NO, p53 can be modulated
  • >1μM NO, it nhibits mitochondrial respiration


Nitric oxide signaling, oxidative stress,  mitochondria, cell damage

Recent data suggests that other NO containing compounds such as S- or N-nitrosoproteins and iron-nitrosyl complexes can be reduced back to produce NO. These NO containing compounds can serve as storage and can reach distant tissues via blood circulation, remote from their place of origin. Hence NO can have both paracrine and ‘endocrine’ effects.

Intracellularly the oxidants present in the cytosol determine the amount of bioacitivity that NO performs. NO can travel roughly 100 microns from NOS enzymes where it is produced.

NO itself in low concentrations have protective action on mitochondrial signaling of cell death.

The aerobic cell was an advance in evolutionary development, but despite the energetic advantage of using oxygen, the associated toxicity of oxygen abundance required adaptive changes.

Oxidation-reduction reactions that are necessary for catabolic and synthetic reactions, can cumulatively damage the organism associated with cancer, cardiovascular disease, neurodegerative disease, and inflammatory overload.  The normal balance between production of pro-oxidant species and destruction by the antioxidant defenses is upset in favor of overproduction of the toxic species, which leads to oxidative stress and disease.

We reviewed the complex interactions and underlying regulatory balances/imbalances between the mechanism of vasorelaxation and vasoconstriction of vascular endothelium by way of nitric oxide (NO), prostacyclin, in response to oxidative stress and intimal injury.

Nitric oxide has a ubiquitous role in the regulation of glycolysis with a concomitant influence on mitochondrial function. The influence on mitochondrial function that is active in endothelium, platelets, vascular smooth muscle and neural cells and the resulting balance has a role in chronic inflammation, asthma, hypertension, sepsis and cancer.

Potential cytotoxic mediators of endothelial cell (EC) apoptosis include increased formation of reactive oxygen and nitrogen species (ROSRNS) during the atherosclerotic process. Nitric oxide (NO) has a biphasic action on oxidative cell killing with low concentrations protecting against cell death, whereas higher concentrations are cytotoxic.

ROS induces mitochondrial DNA damage in ECs, and this damage is accompanied by a decrease in mitochondrial RNA (mtRNA) transcripts, mitochondrial protein synthesis, and cellular ATP levels.

NO and circulatory diseases

Blood vessels arise from endothelial precursors that are thin, flat cells lining the inside of blood vessels forming a monolayer throughout the circulatory system. ECs are defined by specific cell surface markers that characterize their phenotype.

Scientists at the University of Helsinki, Finland, wanted to find out if there exists a rare vascular endothelial stem cell (VESC) population that is capable of producing very high numbers of endothelial daughter cells, and can lead to neovascular growth in adults.

VESCs discovered that reside at the blood vessel wall endothelium are a small population of CD117+ ECs capable of self-renewal.  These cells are capable of undergoing clonal expansion unlike the surrounding ECs that bear limited proliferating potential. A single VESC cell isolated from the endothelial population was able to generate functional blood vessels.

Among many important roles of Nitric oxide (NO), one of the key actions is to act as a vasodilator and maintain cardiovascular health. Induction of NO is regulated by signals in tissue as well as endothelium.

Chen et. al. (Med. Biol. Eng. Comp., 2011) developed a 3-D model consisting of two branched arterioles and nine capillaries surrounding the vessels. Their model not only takes into account of the 3-D volume, but also branching effects on blood flow.

The model indicates that wall shear stress changes depending upon the distribution of RBC in the microcirculations of blood vessels, lead to differential production of NO along the vascular network.

Endothelial dysfunction, the hallmark of which is reduced activity of endothelial cell derived nitric oxide (NO), is a key factor in developing atherosclerosis and cardiovascular disease. Vascular endothelial cells play a pivotal role in modulation of leukocyte and platelet adherence, thrombogenicity, anticoagulation, and vessel wall contraction and relaxation, so that endothelial dysfunction has become almost a synonym for vascular disease. A single layer of endothelial cells is the only constituent of capillaries, which differ from other vessels, which contain smooth muscle cells and adventitia. Capillaries directly mediate nutritional supply as well as gas exchange within all organs. The failure of the microcirculation leads to tissue apoptosis/necrosis.

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