Archive for the ‘Myocardial adenine nucleotide metabolism’ Category

A new way of moving – Michael Sheetz, James Spudich, Ronald Vale

Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 5.5


J Clin Invest. 2012 Oct 1; 122(10): 3374–3377.

The MBI community congratulates Michael Sheetz upon winning the prestigious Albert Lasker Basic Medical Research Award. Michael Sheetz, Director of the Mechanobiology Institute, Singapore, Distinguished Professor of the Department of Biological Sciences, NUS and William R Kenan, Junior Professor at Columbia University, shares this award with two of his collaborators, James Spudich (Stanford University) and Ronald Vale (University of California, San Francisco). The Award was presented at a ceremony on Friday, September 21, 2012, in New York City.


The Albert Lasker Basic Medical Research Award was given to Prof Sheetz, Prof Spudich and Prof Vale in honor of seminal contributions made in establishing methods to study cytoskeletal motor proteins. These developments paved the way to study molecular motors and enabled the discovery of the motor protein, kinesin. The landmark achievements in deciphering new components of cellular motors, which helped explain how these motors worked, were pivotal in understanding the basic fundamental process of energy conversion within the cell. These have led to explorations of these physiologically relevant molecules, as potential drug targets in a variety of disease conditions.


Many basic cellular functions depend on the directed movement of cytoplasmic organelles, macromolecules, membranes or chromosomes from one place to another within the cell. The transport of this intracellular cargo is achieved by molecular motor proteins, such as myosin and kinesin, which provide force and movement through the conversion of chemical energy (ATP) into mechanical energy. Molecular motor proteins move along scaffolds made of specific protein polymers, with kinesins moving along microtubules and myosins along actin filaments, in order to carry their cargo to the appropriate destination within the cell.
Subsequently, Sheetz and Spudich worked out an in-vitro method for visualizing actin filaments creeping along myosin coated surfaces, and this system still remains the gold-standard assay for studying myosin movement. With a read-out in hand, many details of the mechanism of action of the motor molecules within the cell were worked out. Michael Sheetz and colleagues, Ronald Vale and Thomas Reese, carried out pivotal experiments that ultimately led to the discovery of kinesins, a novel and hitherto unknown family of motor proteins. These experiments involved the development of an in vitromotility assay, whereby proteins from the cytoplasm of neuronal cells were shown to power the movement of microtubules across the surface of glass coverslips. This technique was found to be a sensitive and rapid assay for testing the activity of kinesin and was adopted by numerous labs following these crucial initial experiments.

For more details of the award winning contribution towards understanding the basics of cellular machinery, please go to and also


Michael Sheetz, along with James Spudich and Ronald Vale strongly believe in an open culture of scientific exchange. ‘The most interesting scientific insights result from collaborative, interdisciplinary adventures’, has been the one common theme of Michael Sheetz career. A firm believer of an open laboratory concept where students from different labs and backgrounds, share bench space and often ideas, he emulated the Open Lab model (learn more about MBI’s open labs) at the Mechanobiology Institute, Singapore. This new model of open laboratory environment in interdisciplinary institutes provides an excellent way to encourage fast paced discovery process.

My greatest excitement comes from considering the puzzle provided by an unexpected result when new technology is applied to an old problem, says Professor Sheetz.

In his acceptance essay, which can be read here (PDF), Michael Sheetz refers to the importance of collaborations, where the parties are learning from each other and also ‘encourages young scientists to perform speculative experiments whenever they have such an idea, even if most of them fail; since an experiment, even a flawed on, can reveal the solution to an important problem’.


The Mechanobiology Institute is delighted to announce that Michael Sheetz has been selected as a Massry Prize Laureate for 2013.

michaelSheetz_WB_9079Shared with James Spudich (Stanford University) and Ronald Vale (University of California, San Francisco), the award to the trio is a recognition of their work defining molecular mechanisms of ‘intracellular motility.’

This process involves the deployment of molecular machines to move cargo on molecular tracks which are a part of the cellular skeleton.

The discovery of a novel family of motor proteins, the kinesins, by Michael Sheetz, Ronald Vale and Thomas Reese and the methodology developed for the same, proved to be pivotal and the technique developed led to many further discoveries by different laboratories.

Subsequently, Sheetz and Spudich worked out an in-vitro method for visualizing actin filaments creeping along myosin coated surfaces, and this system still remains the gold-standard assay for studying myosin movement. With a read-out in hand, many details of the mechanism of action of the motor molecules within the cell were worked out.







The Lasker Awards: motors take centre stage

Nature Cell Biology | Editorial
Nature Cell Biology 14,1113(2012)

Michael Sheetz, James Spudich and Ronald Vale have now joined the list of Lasker laureates, having jointly received the 2012 Albert Lasker Basic Medical Research Award for their “discoveries concerning cytoskeletal motor proteins, machines that move cargoes within cells, contract muscles, and enable cell movements”1.

Although the mechanism of action and the cellular functions performed by force-generating cytoskeletal motors, including their roles in intracellular trafficking, cell migration, cell division and muscle contraction, are now a fundamental part of cell biology, in the 1970s and 1980s they were still mostly a mystery. Following a postdoctoral fellowship under the guidance of Hugh Huxley, a pioneer of muscle contraction studies, James Spudich established his independent work at the University of California San Francisco (UCSF) and later at Stanford University on what was, at the time, the largely unchartered territory of myosin activity and function. A fortuitous crossing of paths occurred in 1982, when Michael Sheetz joined the Spudich laboratory on sabbatical from his own independent research at the University of Connecticut Health Center. Working together, Spudich and Sheetz demonstrated myosin movement on actin filaments using the Nitella axillaris alga as a model, and later established an in vitro reconstitution system that demonstrated the ability of purified myosin to move on purified actin filaments in the presence of adenosine triphosphate at rates consistent with those of muscle contraction. This seminal work was published in Nature in 19832 and 19853.

Spurred by the exciting work on myosin-based movement, Ronald Vale, then a graduate student at Stanford University, decided with Michael Sheetz to define the particle movement observed in squid axons. Their experiments at the Marine Biology Laboratory in Woods Hole led to a series of groundbreaking Cell publications in 19854, 5, 6, 7, 8, which determined that axonal movement was not driven by myosin on actin filaments as they had anticipated, but was instead occurring on microtubules and was powered by a then-uncharacterized factor that they purified and named kinesin.

These initial efforts investigating myosin- and kinesin-powered motility, and the in vitro assays developed to characterize cytoskeletal motor activities, opened up new and fascinating avenues of research and have become a corner-stone of cell biology today. Following these key discoveries, Spudich went on to define many other aspects of myosin activity and function. Vale continued his work on molecular motors and their cellular roles in his independent research at UCSF, and Sheetz followed a varied research career ranging from motility studies to work on cell adhesion and mechanosensing at Columbia University and the Mechanobiology Institute in Singapore.

In honouring the early work of Sheetz, Spudich and Vale, the Lasker Foundation recognizes the significance of the cytoskeletal motor field in biology, and also the importance of understanding the principles underlying cellular motor function in human diseases in which such activities are deregulated. Indeed, the characterization of normal myosin and kinesin activity and function has served as the spring-board for studies on their impaired or aberrant action in disease, with the goal of developing therapies for heart conditions in the case of myosins, and neurological disorders and malignancy in the case of kinesins.

It should also be noted that the discoveries acknowledged by the Lasker Award and the subsequent scientific careers of the three awardees were the outcome of an inspired mix of cell and molecular biology, biochemistry and physics, among other disciplines, and are thus a testament to the importance of fostering multidisciplinary science. Moreover, as the three award recipients eloquently noted in their Lasker Award acceptance remarks, the motivating force during the exciting times of their initial research on motors was not only a thirst for discovery and a passion for science, but also a strong collaborative spirit. As a fundamentally creative and adventurous endeavour, science is often seen by outsiders as a solitary pursuit of inquiry and testing one’s own ideas. However, the reality of a bustling laboratory reveals that teamwork, discussion and brainstorming, and a successful combination of different personalities, are just as important as individual intellect and drive. In that respect, the dedication, creativity and collaborative efforts of Sheetz, Spudich and Vale should be an inspiration to scientists everywhere.


  2. Sheetz, M. P. & Spudich, J. A. Nature 303, 3135 (1983).
  3. Spudish, J. A., Kron, S. J. & Sheetz, M. P. Nature 315, 584586 (1985).
  4. Vale, R. D., Schnapp, B. J., Reese, T. S. & Sheetz, M. P. Cell 40, 449454 (1985).

One path to understanding energy transduction in biological systems

James A Spudich

Who is not fascinated by the myriad biological movements that define life? From cell migration, cell division and a network of translocation activities within cells to highly specialized muscle contraction, molecular motors operate by burning ATP as fuel. Three types of molecular motors—myosin, kinesin and dynein— and nearly 100 different subtypes transduce that chemical energy into mechanical movements to carry out a wide variety of cellular tasks. Understanding the molecular basis of energy transduction by these motors has taken decades. Our understanding of molecular motors could be viewed as beginning with the two 1954 papers in Nature by Hugh Huxley and Jean Hanson and Andrew Huxley and Rolf Niedergerke, respectively, where the authors proposed the sliding-filament theory of muscle contraction. But a good place to start my story is 1969, when Hugh Huxley, on the basis of his remarkable X-ray diffraction experiments on live muscle coupled with electron microscopy, postulated the swinging crossbridge hypothesis of muscle contraction1. Thus, more than 40 years ago, he proposed the basic concepts of how the myosin molecule produces the sliding of actin filaments to produce contraction. Hugh Huxley laid the foundation for the molecular motor field, and we are all indebted to him. My beginnings in myosin research began as a postdoctoral fellow in Hugh’s laboratory at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England, coincidentally in 1969. But my fascination with science began much earlier.

Neither of my parents was college educated, but they both had keen intellects, positive and enthusiastic outlooks and profound work ethics. My father was intrigued by how things work and shared that interest with my brother John and me. After the coal mines closed, my father taught himself electrical engineering, founded the Spudich Electric Company and patented one of his inventions. He often told John and me, “do whatever excites you, but do it well and be respectful of people you interact with.”

I was captivated with chemistry from a young age. Beginning at the age of six, I mastered every chemistry set I could get. The myriad chemical reactions that could be created using everyday materials, sometimes with marvelously explosive results, fed my excitement for chemistry. It was a world unfamiliar to my parents, but they respected my preoccupations and cleared the pantry of our modest home for me to set up a lab with discarded equipment given to me by my high school chemistry teacher Robert Brandsmark. My brother John has also followed the allure of science into an exciting and distinguished career in basic research. His work has established the molecular basis of signaling in an important class of rhodopsins that he discovered in 1982 (ref. 2). John was my first collaborator.

A chance encounter with Woody Hastings at the University of Illinois launched my experimental-science career. Throughout my undergraduate years, I worked with Woody on bioluminescence in Vibrio fischeri3. I was inspired by his high-spirited fascination with biology and was fortunate to be invited to help him teach in the physiology course at the Marine Biological Laboratory (MBL) in Woods Hole (Fig. 1). At the MBL, I was introduced to the breadth and potential of many biological systems, including muscle contraction.

In 1963 I joined the PhD program in the new Department of Biochemistry at Stanford University, founded by Arthur Kornberg. One of the many remarkable aspects of the biochemistry department was that, although Arthur was my thesis advisor, all the faculty members were my mentors. This unique environment shaped the way I do research and taught me how to be a responsible colleague and a mentor to others (Fig. 2). I learned how important it is to reduce complex biological systems to their essential components and create quantitative in vitro assays for the function of interest. Those years also made it clear to me that interdisciplinary approaches would be key to understanding complex biological processes. So I decided to do postdoctoral work in both genetics and structural biology. I spent one year at Stanford with another influential role model, Charley Yanofsky, working on the genetics of the Escherichia coli tryptophan operon. I then joined Hugh Huxley’s laboratory in Cambridge.

I chose to study the  unanswered questions in cell biology at the time when I established my own laboratory – how the chemical energy of ATP hydrolysis brings about mechanical movement and what roles a myosin-like motor might have in nonmuscle cells.

The essential first steps were to develop a quantitative in vitro motility assay for myosin movement on actin, which is crucial for understanding the molecular mechanism of energy transduction by this system, and to develop a model organism to unravel the molecular basis of the myriad nonmuscle-cell movements that are apparent by light microscopy. We explored Neurospora crassa, Saccharomyces cerevisiae, Physarum polycephalum, Dictyostelium discoideum, Nitella axillaris and other organisms, all unfamiliar to me at the time. The giant cells of the alga Nitella were particularly intriguing because of their striking intracellular cytoplasmic streaming that was visible under a simple light microscope. Although not suitable for biochemistry or genetics, Nitella would assume an important role in my lab a decade later, after Yolande Kersey in Norm Wessells’s laboratory in the Department of Biological Sciences at Stanford showed oriented actin cables lying along chloroplast rows in these cells 5. The slime mold Dictyostelium proved best for our initial biochemical approach 6. Margaret Clarke, my postdoctoral fellow, identified a myosin in Dictyostelium. We also showed that actin is associated with the cell membrane in this organism, and we isolated membranecoated polystyrene beads with actin filaments emanating from them. We were tremendously excited about the possibilities these results presented as a small step along the way to an in vitro motility assay where these actin-coated particles could move along a myosin-coated surface (Fig. 3).

Figure 3 Dictyostelium has a muscle-like myosin and membrane-associated actin. (a) A possible scheme for pulling two membranes together (redrawn from ref. 6). (b) Margaret Clarke discovered myosin II in Dictyostelium and showed that it forms bipolar thick filaments, similar to muscle myosin. (c) Phagocytized polystyrene beads offered an opportunity to explore one version of an in vitro motility assay where the beads may be pulled along by myosin. Taken from my laboratory notebook, 21 January 1973.

Figure 4 One approach to an in vitro motility assay from a totally defined system. (a) The concept was to observe myosin-coated beads moving along fixed actin filaments oriented by buffer flow. The actin filaments had biotinylated severin bound to their barbed ends; the barbed ends were attached to an avidin-coated surface by way of the tight avidin-biotin link. The filaments were oriented by buffer flow. B, biotin; S, severin. (b) Myosin-coated beads were observed by light microscopy to move upstream toward the barbed end of the surface-attached actin filaments. The position of each of the three bead aggregates is shown as a function of time. This was the first demonstration of quantitative, directed movement of myosin along actin with a totally defined system (taken from ref. 11). ATP binding releases the myosin Myosin binds to actin ATP ADP Pi ADP .

In 1977 I joined the Department of Structural Biology at Stanford. In the next years we extensively characterized the actin-myosin system in Dictyostelium. My student Arturo De Lozanne made the chance discovery that genes in Dictyostelium can be knocked out by homologous recombination and provided the first genetic proof that myosin II is essential for time that myosin II drove the forward movement of cells. Dietmar Manstein, Meg Titus and Arturo then extended these experiments to create a myosin-null cell8, which was crucial to our later work using mutational analysis to define the structure-function relationships of the myosin molecule and for important experiments in support of the swinging cross-bridge hypothesis9. Interestingly, reports from a number of laboratories between 1969 and 1980 did not support the swinging cross-bridge model, and it was more imperative than ever to develop a quantitative in vitro motility system to test the various models under consideration. In 1981 we identified and purified Dictyostelium severin, a protein that tightly binds the ‘barbed ends’ of actin filaments. This provided an opportunity to try another version of an in vitro motility assay. Using biotinylated severin, we attached the actin filament barbed ends to an avidin-coated slide and flowed aqueous solution over them. Long filaments attached to the surface at one end would be expected to orient in the direction of the flowing solution (Fig. 4a). We placed myosin-coated beads on these actin-coated slides and added ATP but saw only sporadic movements. In retrospect, we probably did not have sufficient alignment of filaments; we were not monitoring filament alignment at that time by electron microscopy, as we did later.

A key breakthrough occurred in 1982 when Mike Sheetz came to my laboratory on sabbatical. Not certain what component of our system might be limiting our approach, we took advantage of the known orientation of actin filaments in Nitella5 to overcome the actin filament alignment problem. Peter Sargent, a neurobiologist in the Structural Biology Department at that time, helped us cut open a Nitella cell, and we attached it to a surface to expose the actin fibers. We added myosin-coated beads and eureka! We saw robust ATP-dependent unidirectional movement along chloroplast rows, which mark the actin fibers 10.

Armed with the Nitella results, Mike left my lab and went to the MBL to explore whether myosin-coated vesicles may account for the particle movements observed in squid axons. Ron Vale, then a graduate student at Stanford with Eric Shooter, was fascinated by the movement of organelles in nerve axons and joined Mike at the MBL. To their great surprise, they found that movement in axons is not myosin driven. Instead, they discovered the new molecular motor kinesin, a discovery that completely energized the field and opened up years of exciting work from their laboratories and many others. ..


The combination of the in vitro motility assay and the Dictyostelium myosin-null cell provided powerful tools for Kathy Ruppel, Taro Uyeda, Dietmar Manstein, William Shih, Coleen Murphy, Meg Titus, Tom Egelhoff and others in my lab to use mutations along myosin to define the biochemical, biophysical and assembly properties of the molecule. Our results were consistent with the proposed actin-activated myosin chemomechanical cycle derived largely from the elegant biochemical kinetic studies from Edward Taylor’s laboratory in the early 1970’s (ref. 15) (Fig. 5). Then, in 1993, Ivan Rayment and his colleagues16 obtained a high-resolution crystal structure of myosin S1. Ivan’s pivotal work allowed us to place our mutational analyses in a myosin structure-function context.

Figure 5 The actin-activated myosin chemomechanical cycle. This cycle, extensively studied by many researchers over several decades, was derived from kinetic studies of Lymn and Taylor 15. A mechanical stroke only occurs when the myosin is strongly bound to actin. Our mutational analyses of Dictyostelium myosin II probed each of the steps shown and provided structure-function analyses that helped define how the myosin motor works. ADP-Pi , ADP and inorganic phosphate, the products of ATP hydrolysis, remain bound to the active site until actin binds to the myosin.

Figure 6 In vitro motility taken to the single-molecule level using the physics of laser trapping. (a) The Kron in vitro motility assay observing fluorescent actin filaments (yellow) moving on a myosin-coated (red) surface. (b) Two polystyrene beads attached to the ends of a single actin filament are trapped in space by laser beams. The filament is lowered onto a single myosin molecule on a bump on the surface (gray sphere). (c) Jeff Finer building the dual-beam laser trap in around 1990.

Fundamental issues still remained— primarily to establish the step size that the myosin takes for each ATP hydrolysis, which was under considerable debate.


One of my great satisfactions is that the more detailed understanding of energy transduction by myosin has led to potential clinical therapies. A small molecule that binds and activates b-cardiac myosin is now in clinical trials for the treatment of heart failure, and another small molecule currently in clinical trials activates skeletal muscle contraction and may aid patients with amyotropic lateral sclerosis and other diseases.



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Introduction to Metabolomics

Introduction to Metabolomics

Author: Larry H. Bernstein, MD, FCAP


This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time bachalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career.

In the Preface, I failed to disclose that the term Metabolomics applies to plants, animals, bacteria, and both prokaryotes and eukaryotes.  The metabolome for each organism is unique, but from an evolutionary perspective has metabolic pathways in common, and expressed in concert with the environment that these living creatures exist. The metabolome of each has adaptive accommodation with suppression and activation of pathways that are functional and necessary in balance, for its existence.  Was it William Faulkner who said in his Nobel Prize acceptance that mankind shall not merely exist, but survive? That seems to be the overlying theme for all of life. If life cannot persist, a surviving “remnant” might continue. The history of life may well be etched into the genetic code, some of which is not expressed.

This work is apportioned into chapters in a sequence that is first directed at the major sources for the energy and the structure of life, in the carbohydrates, lipids, and fats, which are sourced from both plants and animals, and depending on their balance, results in an equilibrium, and a disequilibrium we refer to as disease.  There is also a need to consider the nonorganic essentials which are derived from the soil, from water, and from the energy of the sun and the air we breathe, or in the case of water-bound metabolomes, dissolved gases.

In addition to the basic essential nutrients and their metabolic utilization, they are under cellular metabolic regulation that is tied to signaling pathways.  In addition, the genetic expression of the organism is under regulatory control by the interaction of RNAs that interact with the chromatin genetic framework, with exosomes, and with protein modulators.This is referred to as epigenetics, but there are also drivers of metabolism that are shaped by the interactions between enzymes and substartes, and are related to the tertiary structure of a protein.  The framework for diseases in a separate chapter.  Pharmaceutical interventions that are designed to modulate specific metabolic targets are addressed as the pathways are unfolded. Neutraceuticals and plant based nutrition are covered in Chapter 8.

Chapter 1: Metabolic Pathways

Chapter 2. Lipid Metabolism

Chapter 3. Cell Signaling

Chapter 4. Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8. Impairments in Pathological States: Endocrine Disorders; Stress Hypermetabolism and Cancer

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Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation: a Compilation of Articles in the Journal

Compilation of References by Leaders in Pharmaceutical Business Intelligence in the Journal about
Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation

Curator: Larry H Bernstein, MD, FCAP


  1. The Human Proteome Map Completed

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

  1. Proteomics – The Pathway to Understanding and Decision-making in Medicine

Author and Curator, Larry H Bernstein, MD, FCAP

3. Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets

Author and Curator, Larry H Bernstein, MD, FCAP         of-therapeutic-targets/

  1. Expanding the Genetic Alphabet and Linking the Genome to the Metabolome

Author and Curator, Larry H Bernstein, MD, FCAP                metabolome/

5. Genomics, Proteomics and standards

Larry H Bernstein, MD, FCAP, Author and Curator

6. Proteins and cellular adaptation to stress

Larry H Bernstein, MD, FCAP, Author and Curator



  1. Extracellular evaluation of intracellular flux in yeast cells

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

  1. Metabolomic analysis of two leukemia cell lines. I.

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

  1. Metabolomic analysis of two leukemia cell lines. II.

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

  1. Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics

Reviewer and Curator, Larry H. Bernstein, MD, FCAP          in-nutritional-metabolism-and-biotherapeutics/

  1. Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation

Larry H. Bernstein, MD, FCAP, Reviewer and curator              metabolism-provides-homeomeostatic-regulation/

Metabolic Pathways

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

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

  1. Mitochondria: More than just the “powerhouse of the cell”

Ritu Saxena, PhD

  1. Mitochondrial fission and fusion: potential therapeutic targets?

Ritu saxena

4.  Mitochondrial mutation analysis might be “1-step” away

Ritu Saxena

  1. Selected References to Signaling and Metabolic Pathways in

Curator: Larry H. Bernstein, MD, FCAP                     leaders-in-pharmaceutical-intelligence/

  1. Metabolic drivers in aggressive brain tumors

Prabodh Kandal, PhD

  1. Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes

Writer and Curator, Aviva Lev-Ari, PhD, RD                        information-genetic-association-links-unknown-metabolites-to-functionally-related-genes/

  1. Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation

Larry H Bernstein, MD, FCAP, author and curator            glycolysis-metabolic-adaptation/

  1. Therapeutic Targets for Diabetes and Related Metabolic Disorders

Reporter, Aviva Lev-Ari, PhD, RD

10.  Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation

Larry H. Bernstein, MD, FCAP, Reviewer and curator              metabolism-provides-homeomeostatic-regulation/

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

Larry H. Bernstein, MD, FCAP, Curator:                          exchange-energy/

12. Studies of Respiration Lead to Acetyl CoA

13. Lipid Metabolism

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

14. Carbohydrate Metabolism

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

15. Update on mitochondrial function, respiration, and associated disorders

Larry H. Bernstein, MD, FCAP, Author and Curator                   disorders/

16. Prologue to Cancer – e-book Volume One – Where are we in this journey?

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

17. Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H. Bernstein, MD, FCAP          how-we-got-here/

18. Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K

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

19. The Binding of Oligonucleotides in DNA and 3-D Lattice Structures

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

20. Mitochondrial Metabolism and Cardiac Function

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

21. How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia

Curator: Larry H. Bernstein, MD, FCAP

22. AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo

Author and Curator: Stephen J. Williams, PhD         tumor-growth-in-vivo/

23. A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

Author and Curator: Larry H. Bernstein, MD, FCAP                         conundrum/

24. Mitochondrial Damage and Repair under Oxidative Stress

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

25. Nitric Oxide and Immune Responses: Part 2

Author and Curator: Aviral Vatsa, PhD, MBBS

26. Overview of Posttranslational Modification (PTM)

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

27. Malnutrition in India, high newborn death rate and stunting of children age under five years

Writer and Curator: Larry H. Bernstein, MD, FCAP                   children-age-under-five-years/

28. Update on mitochondrial function, respiration, and associated disorders

Writer and Curator: Larry H. Bernstein, MD, FCAP                  disorders/

29. Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease

Larry H. Bernstein, MD, FCAP, Curator         in-renal-disease/

30. Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine

Larry H. Bernstein, MD, FCAP, writer, and Aviva Lev- Ari, PhD, RN                                  translational_medicine-part_2/

31. Epilogue: Envisioning New Insights in Cancer Translational Biology
Series C: e-Books on Cancer & Oncology

Author & Curator: Larry H. Bernstein, MD, FCAP, Series C Content Consultant

32. Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone                         and Neurotransmitter

Writer and Curator: Larry H Bernstein, MD, FCAP and
Curator and Content Editor: Aviva Lev-Ari, PhD, RN                    hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocy

33. Cardiac Contractility & Myocardial Performance: Therapeutic Implications of Ryanopathy (Calcium Release-                           related Contractile Dysfunction) and Catecholamine Responses

Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
Author and Curator: Larry H Bernstein, MD, FCAP
and Article Curator: Aviva Lev-Ari, PhD, RN      and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-                    contractile/

34. Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Author and Curator: Larry H Bernstein, MD, FCAP Author: Stephen Williams, PhD, and Curator: Aviva Lev-Ari, PhD, RN

35. Identification of Biomarkers that are Related to the Actin Cytoskeleton

Larry H Bernstein, MD, FCAP, Author and Curator                           cytoskeleton/

36. Advanced Topics in Sepsis and the Cardiovascular System at its End Stage

Author: Larry H Bernstein, MD, FCAP              End-Stage/

37. The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology

Demet Sag, PhD, Author and Curator               immunology/

38. IDO for Commitment of a Life Time: The Origins and Mechanisms of IDO, indolamine 2, 3-dioxygenase

Demet Sag, PhD, Author and Curator             ido-indolamine-2-3-dioxygenase/

39. Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Homeostasis of Immune Responses for Good and Bad

Curator: Demet Sag, PhD, CRA, GCP           of-immune-responses-for-good-and-bad/

40. Signaling Pathway that Makes Young Neurons Connect was discovered @ Scripps Research Institute

Reporter: Aviva Lev-Ari, PhD, RN                     discovered-scripps-research-institute/

41. Naked Mole Rats Cancer-Free

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

42. Late Onset of Alzheimer’s Disease and One-carbon Metabolism

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

43. Problems of vegetarianism

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

44.  Amyloidosis with Cardiomyopathy

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

45. Liver endoplasmic reticulum stress and hepatosteatosis

Larry H Bernstein, MD, FACP

46. The Molecular Biology of Renal Disorders: Nitric Oxide – Part III

Curator and Author: Larry H Bernstein, MD, FACP

47. Nitric Oxide Function in Coagulation – Part II

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

48. Nitric Oxide, Platelets, Endothelium and Hemostasis

Curator and Author: Larry H Bernstein, MD, FACP

49. Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Curator and Author: Larry H Bernstein, MD, FACP

50. Nitric Oxide and Immune Responses: Part 1

Curator and Author:  Aviral Vatsa PhD, MBBS

51. Nitric Oxide and Immune Responses: Part 2

Curator and Author:  Aviral Vatsa PhD, MBBS

52. Mitochondrial Damage and Repair under Oxidative Stress

Curator and Author: Larry H Bernstein, MD, FACP

53. Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?

Curator and Author: Larry H Bernstein, MD, FACP                 century-view/

54. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Curator and Author: Larry H Bernstein, MD, FACP                  proteolysis-and-cell-apoptosis/

55. Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III

Curator and Author: Larry H Bernstein, MD, FACP                   proteolysis-and-cell-apoptosis-reconsidered/

56. Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II

Curator and Author: Larry H Bernstein, MD, FACP

57. New Insights on Nitric Oxide donors – Part IV

Curator and Author: Larry H Bernstein, MD, FACP

58. Crucial role of Nitric Oxide in Cancer

Curator and Author: Ritu Saxena, Ph.D.

59. Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

Curator and Author: Larry H Bernstein, MD, FACP         a-concomitant-influence-on-mitochondrial-function/

60. Targeting Mitochondrial-bound Hexokinase for Cancer Therapy

Curator and Author: Ziv Raviv, PhD, RN 04/06/2013

61. Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I

Curator and Author: Larry H Bernstein, MD, FACP

Genomics, Transcriptomics, and Epigenetics

  1. What is the meaning of so many RNAs?

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

  1. RNA and the transcription the genetic code

Larry H. Bernstein, MD, FCAP, Writer and Curator

  1. A Primer on DNA and DNA Replication

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

4. Synthesizing Synthetic Biology: PLOS Collections

Reporter: Aviva Lev-Ari

5. Pathology Emergence in the 21st Century

Author and Curator: Larry Bernstein, MD, FCAP

6. RNA and the transcription the genetic code

Writer and Curator, Larry H. Bernstein, MD, FCAP

7. A Great University engaged in Drug Discovery: University of Pittsburgh

Larry H. Bernstein, MD, FCAP, Reporter and Curator

8. microRNA called miRNA-142 involved in the process by which the immature cells in the bone  marrow give                              rise to all the types of blood cells, including immune cells and the oxygen-bearing red blood cells

Aviva Lev-Ari, PhD, RN, Author and Curator                   immature-cells-in-the-bone-marrow-give-rise-to-all-the-types-of-blood-cells-including-immune-cells-and-the-oxygen-             bearing-red-blood-cells/

9. Genes, proteomes, and their interaction

Larry H. Bernstein, MD, FCAP, Writer and Curator

10. Regulation of somatic stem cell Function

Larry H. Bernstein, MD, FCAP, Writer and Curator    Aviva Lev-Ari, PhD, RN, Curator

11. Scientists discover that pluripotency factor NANOG is also active in adult organisms

Larry H. Bernstein, MD, FCAP, Reporter           adult-organisms/

12. Bzzz! Are fruitflies like us?

Larry H Bernstein, MD, FCAP, Author and Curator

13. Long Non-coding RNAs Can Encode Proteins After All

Larry H Bernstein, MD, FCAP, Reporter

14. Michael Snyder @Stanford University sequenced the lymphoblastoid transcriptomes and developed an
allele-specific full-length transcriptome

Aviva Lev-Ari, PhD, RN, Author and Curator            transcriptomes-and-developed-an-allele-specific-full-length-transcriptome/

15. Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: Views by Larry H                                     Bernstein, MD, FCAP

Author: Larry H Bernstein, MD, FCAP                        cardiovascular-disease-views-by-larry-h-bernstein-md-fcap/

16. Observations on Finding the Genetic Links in Common Disease: Whole Genomic Sequencing Studies

Author an curator: Larry H Bernstein, MD, FCAP

17. Silencing Cancers with Synthetic siRNAs

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

18. Cardiometabolic Syndrome and the Genetics of Hypertension: The Neuroendocrine Transcriptome Control Points

Reporter: Aviva Lev-Ari, PhD, RN

19. Developments in the Genomics and Proteomics of Type 2 Diabetes Mellitus and Treatment Targets

Larry H. Bernstein, MD, FCAP, Reviewer and Curator           mellitus-and-treatment-targets/

20. Loss of normal growth regulation

Larry H Bernstein, MD, FCAP, Curator

21. CT Angiography & TrueVision™ Metabolomics (Genomic Phenotyping) for new Therapeutic Targets to Atherosclerosis

Reporter: Aviva Lev-Ari, PhD, RN           new-therapeutic-targets-to-atherosclerosis/

22.  CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics

Genomics Curator, Larry H Bernstein, MD, FCAP                      computational-genomics/

23. Big Data in Genomic Medicine

Author and Curator, Larry H Bernstein, MD, FCAP

24. From Genomics of Microorganisms to Translational Medicine

Author and Curator: Demet Sag, PhD                      microorganisms-to-translational-medicine/

25. Summary of Genomics and Medicine: Role in Cardiovascular Diseases

Author and Curator, Larry H Bernstein, MD, FCAP

 26. Genomic Promise for Neurodegenerative Diseases, Dementias, Autism Spectrum, Schizophrenia, and Serious                      Depression

Author and Curator, Larry H Bernstein, MD, FCAP        spectrum-schizophrenia-and-serious-depression/

 27.  BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair

Sudipta Saha, PhD         in-transcription-ubiquitination-and-dna-repair/

28. Personalized medicine gearing up to tackle cancer

Ritu Saxena, PhD

29. Differentiation Therapy – Epigenetics Tackles Solid Tumors

Stephen J Williams, PhD

30. Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment

     Aviva Lev-Ari, PhD, RN          detection-treatment/

31. The Molecular pathology of Breast Cancer Progression

Tilde Barliya, PhD

32. Gastric Cancer: Whole-genome reconstruction and mutational signatures

Aviva Lev-Ari, PhD, RN                   signatures-2/

33. Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine –                                                       Part 1 (

Aviva  Lev-Ari, PhD, RN

34. LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer                                         Personalized Treatment: Part 2

A Lev-Ari, PhD, RN       drug-selection-in-cancer-personalized-treatment-part-2/

35. Personalized Medicine: An Institute Profile – Coriell Institute for Medical Research: Part 3

Aviva Lev-Ari, PhD, RN        research-part-3/

36. Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of                           Cancer Scientific Leaders @

Aviva Lev-Ari, PhD, RN Cancer_Management-      Prospects_of_Prevention_and_Cure/

37.  GSK for Personalized Medicine using Cancer Drugs needs Alacris systems biology model to determine the in silico
effect of the inhibitor in its “virtual clinical trial”

Aviva Lev-Ari, PhD, RN             systems-biology-model-to-determine-the-in-silico-effect-of-the-inhibitor-in-its-virtual-clinical-trial/

38. Personalized medicine-based cure for cancer might not be far away

Ritu Saxena, PhD

39. Human Variome Project: encyclopedic catalog of sequence variants indexed to the human genome sequence

Aviva Lev-Ari, PhD, RN         indexed-to-the-human-genome-sequence/

40. Inspiration From Dr. Maureen Cronin’s Achievements in Applying Genomic Sequencing to Cancer Diagnostics

Aviva Lev-Ari, PhD, RN                genomic-sequencing-to-cancer-diagnostics/

41. The “Cancer establishments” examined by James Watson, co-discoverer of DNA w/Crick, 4/1953

Aviva Lev-Ari, PhD, RN         of-dna-wcrick-41953/

42. What can we expect of tumor therapeutic response?

Author and curator: Larry H Bernstein, MD, FACP

43. Directions for genomics in personalized medicine

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

44. How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis.

Stephen J Williams, PhD            mediated-tumorigenesis/

45. mRNA interference with cancer expression

Author and Curator, Larry H. Bernstein, MD, FCAP

46. Expanding the Genetic Alphabet and linking the genome to the metabolome

Aviva Lev-Ari, PhD, RD               metabolome/

47. Breast Cancer, drug resistance, and biopharmaceutical targets

Author and Curator: Larry H Bernstein, MD, FCAP

48.  Breast Cancer: Genomic profiling to predict Survival: Combination of Histopathology and Gene Expression                            Analysis

Aviva Lev-Ari, PhD, RD           histopathology-and-gene-expression-analysis

49. Gastric Cancer: Whole-genome reconstruction and mutational signatures

Aviva  Lev-Ari, PhD, RD                   signatures-2/

50. Genomic Analysis: FLUIDIGM Technology in the Life Science and Agricultural Biotechnology

Aviva Lev-Ari, PhD, RD                   agricultural-biotechnology/

51. 2013 Genomics: The Era Beyond the Sequencing Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

Aviva Lev-Ari, PhD, RD

52. Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1

Aviva Lev-Ari, PhD, RD Shift in Human Genomics_/

Signaling Pathways

  1. Proteins and cellular adaptation to stress

Larry H Bernstein, MD, FCAP, Curator

  1. A Synthesis of the Beauty and Complexity of How We View Cancer:
    Cancer Volume One – Summary

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

  1. Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in
    serous endometrial tumors

Sudipta Saha, PhD           ligase-complex-genes-in-serous-endometrial-tumors/

4.  Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition

Stephen J Williams, PhD              transition-in-prostate-cancer-cells/

5. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Author and Curator: Larry H Bernstein, MD, FCAP                   proteolysis-and-cell-apoptosis/

6. Signaling and Signaling Pathways

Larry H. Bernstein, MD, FCAP, Reporter and Curator

7.  Leptin signaling in mediating the cardiac hypertrophy associated with obesity

Larry H. Bernstein, MD, FCAP, Reporter and Curator            with-obesity/

  1. Sensors and Signaling in Oxidative Stress

Larry H. Bernstein, MD, FCAP, Reporter and Curator

  1. The Final Considerations of the Role of Platelets and Platelet Endothelial Reactions in Atherosclerosis and Novel

Larry H. Bernstein, MD, FCAP, Reporter and Curator                      endothelial-reactions-in-atherosclerosis-and-novel-treatments

10.   Platelets in Translational Research – Part 1

Larry H. Bernstein, MD, FCAP, Reporter and Curator

11.  Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and
Cardiovascular Calcium Signaling Mechanism

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             smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

12. 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       kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-           differen/

13.  Nitric Oxide Signalling Pathways

Aviral Vatsa, PhD, MBBS

14. Immune activation, immunity, antibacterial activity

Larry H. Bernstein, MD, FCAP, Curator

15.  Regulation of somatic stem cell Function

Larry H. Bernstein, MD, FCAP, Writer and Curator    Aviva Lev-Ari, PhD, RN, Curator

16. Scientists discover that pluripotency factor NANOG is also active in adult organisms

Larry H. Bernstein, MD, FCAP, Reporter

Read Full Post »

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 »

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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