Posts Tagged ‘adducts’

English: reaction of the lactate dehydrogenase...

English: reaction of the lactate dehydrogenase: pyruvate (left) is oxidized to lactate (right) by expense of NADH Deutsch: Reaktionsmechanismus der Lactatdehydrogenase: normalerweise wird Pyruvat (links) wird mittels NADH zu Lactat (rechts) oxidiert (Photo credit: Wikipedia)

Remembering a Great Scientist among Mentors

Author: Larry H. Bernstein, MD



N a t h a n O r a m K a p l a n (1917-1986) This is a portrait of my experience and a tribute to a giant of metabolic discovery in the third quarter of the 20th century.

Part I. My interest leading up to my experience with Nathan O. Kaplan

A. Graduate School Experience

Working under Harry Maisel, my teacher and mentor led to a thesis on “The Ontogeny of the Crystalline Proteins of the Bovine Lens.” But while I was carrying out those studies, I also investigated the “Changes in the Isoenzymes of Lactic Dehydrogenase”. This required the use of starch gel electrophoresis introduced by David Poulik and Oliver Smithies, prior to the use of polyacrylamide. The gels were bisected and stained for protein or for enzymatic activity.
There was a controversy at that time over a view held by Elliott Vessell and NO Kaplan’s argument that the LDH is a tetramer composed of M-type and H-type subunits polymerized that have metabolically different roles.

  •  The lens of the eye and circulating mature red cells depend on glycolysis for 86% of their energy, and the reminder is in the pentose phosphate shunt (essential for nucleotide and nucleoside synthesis). Mature lens and red cells have a predominantly H4, H3M pattern, so that LD1 and 2 are elevated in hemolysis and in heart attack. The distribution in kidney is a mixture of the medullary and the cortical patterns, as the cortex has a high rate of aerobic glycolysis, and it has a rich vasculature of glomuli capillaries and arterioles.
  • The cardiomyocyte and skeletal muscle both have nuclei, but one has the H4, H3M and the other has M4, M3H predominant pattern.

So it would appear that Kaplan had a better grasp of the problem. One the one hand, he held that the H-type LD subunit is regulatory, while the M-type is not. Renal cortical epithelial cells have a high rate of aerobic glycolysis, aka, mitochondria. On the other hand, he also saw an organ pattern relationship represented by TPN vs DPN, depending on primary role in synthetic activity or high energy utilization.
Functional lactate dehydrogenase are homo or hetero tetramers composed of M and H protein subunits encoded by the LDHA and LDHB genes, respectively:

  1. LDH-1 (4H)—in the heart
  2. LDH-2 (3H1M)—in the reticuloendothelial system
  3. LDH-3 (2H2M)—in the lungs
  4. LDH-4 (1H3M)—in the kidneys, placenta, and pancreas
  5. LDH-5 (4M)—in the liver and striated muscle[2]

B. Postgraduate Medical Education

In residency in Pathology I came under the influence of the late Masahiro Chiga, a pathologist and noted biochemist who had discovered that myokinase is distinguished from adenylate kinase by inhibition with sulfhydryl reagents. He encouraged me to go the University of California, San Diego, where I could learn from from Nate Kaplan. I completed my postgraduate education on an NIH Training Grant in Cardiovascular Pathology under AA Liebow, work withing with Nathan Kaplan, and well prepared in both scientific method and in Pathology with several published papers.

C. The m-type (mitochondrial) malate dehydrogenase

My main research occupation was in work the two major isoenzymes of malate dehydrogenase (c- and m-type) and the nature of a ternary complex of enzyme, oxaloacetate, and DPN (NAD) formed during the forward reaction of OAA to malate generating DPN from DPNH. The regulatory nature of the c- and m-type MDH in hydrogen transfer between the cytoplasm and mitochondrion were of great interest.

D . A full fledged Pathologist/Clinical Pathologist

My focus was on the mitochondrial and cytoplasmic malate dehydrogenases, in hepatoma, in comparative animal life, and in different tissues when I joined Herschel Sidranski in a Department of Pathology at University of South Florida, Tampa.

Part II. About Nate Kaplan

The following material is extracted from these sources:

  1. A biographical memoir, William D. McElroy (National Academy of Sciences, 1994)
  2. Nathan O. Kaplan Papers, 1943-1986. UCSD, Geisel Library. Mandeville Special Collections Library. La Jolla, California 92093-0175. Collection number: MSS 0099.
  3. QUOTATIONS BY William Allison, Morris Friedkin, Martin Kamen, H. A. Barker, David Greenberg, Mary Ellen Jones, and W. P. Jencks are found in a memorial publication dedicated to Nate, and appeared in Analytical Biochemistry 1987;161:229-44.

A. Early and Predoctoral

Kaplan’s formative work with David Greenberg in the Biochemistry Division of the Berkeley medical school, involved studying phosphate utilization, distribution, and turnover in various nutritional states and required extracting, separating, and identifying organic phosphate compounds of metabolic significance. Martin Kamen wrote a brief account of Nate’s stay at Berkeley: Nate’s collaboration with Michael Doudoroff and William Zev Hassid demonstrated that glucose 1-phosphate and fructose were the products of sucrose breakdown by enzymatic transfer of a glucosyl moiety to radioactive phosphate (from EO Lawrence’s lab).B. Postdoctoral workNate attended a microbial metabolism course given by H. A. Barker and at that time Lipmann’s article on phosphate bond energy appeared in Volume 1 of Advances in Enzymology (1941).

In 1945, he focused on coenzyme A as a research associate with Nobel laureate Fritz Lipmann at the MGH. Lipmann had recently shown that the acetylation of sulfanilamide by pigeon liver extracts required a heat-stable factor, which Kaplan purified, now known as coenzyme A. He was also instrumental in determining its structure, and helped establish the universality of coenzyme A in 2-carbon metabolism. Nate, G. David Novelli and Beverly Guirard, soon found that Coenzyme A contained pantothenic acid, and later Shuster and Kaplan found that a phosphate group was attached to the 3′-hydroxyl of the ribose ring of adenylic acid. In the meantime Kaplan and Lipmann found that most of the pantothenate in tissues was present in coenzyme A. For his contributions to the work on coenzyme A, Nate shared the Nutrition Award in 1948 and received the Eli Lily Award in Biochemistry in 1953.

B. McCollum-Pratt Institute

Kaplan and Sidney Colowick (who had just left the Cori laboratory at Washington University in St. Louis) developed a successful and productive collaboration at the University of Illinois prior to their invitation by W.D. McElroy to the McCollum-Pratt Institute at Johns Hopkins University studying the chemistry of the pyridine nucleotide coenzymes and the enzymes that are involved with them. This collaboration led to the founding in 1955 of the classic series, Colowick and Kaplan’s Methods in Enzymology, which had more than 140 volumes in 1986, and continues today.
McElroy recalls the establishment of the McCollum-Pratt Institute at Johns Hopkins. “There was a large gap between European-English biochemistry and that of the United States. Only in 1941 when Lipmann and Kalckar published their famous reviews was ATP introduced widely in the U.S. biochemical literature. Nate spent hours with Dr. Elmer McCollum learning all he could about the history of nutrition and biochemistry. The year that Warburg discovered the requirement of Mg2+ for the triose phosphate dehydrogenase was the same year that McCollum demonstrated it as an essential micronutrient in animals.
Kaplan and Colowick carried out studies on oxidative phosphorylations in
the microbe Pseudomonas aeruginosa, a well-known bacteria with a very high oxidative capacity. Sid and Nate made the interesting discovery that a transfer of hydrogen from the NADPH to NAD was occurring when both pyridine nucleotides were present in the reaction mixture. It was this discovery that led to many efforts to characterize the transhydrogenase that was obviously present in the system.

Work done by Kaplan, Colowick, and Elizabeth Neufeld at the Institute describes discovery of a transhydrogenase from pig heart mitochondria that transfers hydrogen from TPNH (NADPH) to DPN (NAD), which Martin Klingenberg and Lars Ernster showed transfers electrons from NADH to NADP in an energy-dependent reaction.
(From the McCollum-Pratt Institute, The Johns Hopkins University, Baltim,ore,
(Received for publication, April 15, 1953)

In previous communications, an enzyme from Pseudomonas fluorescens has been described, that catalyzes a transfer of electrons between the pyridine nucleotides (1-3). This enzyme, termed pyridine nucleotide transhydrogenase, was shown to promote Reactions 1 to 5.’
(1) TPNH + DPN+ -+ TPN+ + DPNH
(2) TPNH + desamino DPN+ + TPN+ + desamino DPNH
(3) Desamino TPNH + DPN+ -+ desamino TPN+ + DPNH
(4) Desamino DPNH + DPN+ + desamino DPN+ + DPNH
(5) Desamino TPNH + TPN+ 4 desamino TPN+ + TPNH
We have been able to detect the presence of pyridine nucleotide transhydrogenase activity in a number of animal tissues. The present paper deals with the properties and specificity of the animal transhydrogenases, and indicates differences between the animal and bacterial systems.


McElroy had a number of nitrate mutants in Neurospora that could reduce nitrate to nitrite, but the latter would not be further metabolized. Nate had some FAD which led to the eventual discovery that reduced FAD was the immediate electron donor for the reduction of molybdenum and subsequently the reduction of nitrate.
In addition, Nate and Sid found an enzyme which could split NAD at the nicotinamide ribosidic linkage present in relatively large amounts in zinc-deficient Neurospora. The compound was the alpha isomer of NAD, which exhibited very low or no activity with most dehydrogenases. When Nate and Sid studied NADase isolated from mammalian sources they found that the animal enzyme was inhibited by nicotinamides whereas the Neurospora enzyme was quite insensitive to the free vitamin. Leonard Zatman observed that radioactive nicotinamide could be incorporated into NAD and demonstrated that an exchange reaction was occurring.

Following the discovery of Neurospora DPNase, Nate and Sid worked together on enzymes concerned with DPN, particularly the exchange reactions involving ADP ribosyl enzyme and various nicotinamide derivatives. The work on NAD and NADP and the analogs, which they were able to make by the exchange reaction, formed the basis for an intense collaboration between Nate and Colowick concerning the function of these coenzymes in various dehydrogenases.

Using the exchange reaction, Nate was able to prepare the acetylpyridine derivative of NAD, which turned out to be extremely important as it was the compound used later by Nate to compare the biochemistry of various dehydrogenases. The ratio of the activity with DPN and the acetylpyridine analog was a very sensitive measure of the differences of various dehydrogenases in different species and in different organs. The reduced form of the acetylpyridine NAD had an absorption maximum at 375 nm as compared to 340 nm for NADH. This, of course, eventually led to the important research on isozymes.

He studied the isozymes of various dehydrogenases and noted their changes during development. Probably his best-known work in the area was concerned with the M and H isozymes of lactic dehydrogenase, this latter work leading to his interest in cancer metabolism. One of Nate’s great assets was willingness to help anyone in need—graduate students, postdocs, faculty and visiting scientists. While no mention is made of this he brought on a bright high school student, Francis Stolzenbach, who would stay with him for over 20 years.

C. Brandeis University

He established the Graduate Department of Biochemistry at Brandeis University in 1957, in association with Martin Kamen who joined him at Brandeis, and hired carefully selected young assistant professors. Mr. Rosenstiel, a rare individual who preferred to “buy brains, not bricks”, gave $1,000,000 to start the department and supplemented this later with additional support to the department and university. Kaplan and Kamen were able to turn this investment into a 2,300 percent profit from various sources, to provide a strong base of support for the department. The scientific productivity of this fledling department was of a caliber that it gained international recognition in a very short time. Brandeis had only been founded in 1948, and became a major, research oriented university in the sciences in the 1960s.
When he moved to Brandeis, Nate was able to distinguish the heart and muscle lactate dehydrogenase of a given species using NAD analogs. He found that the heart enzyme of one species was much more closely related to the heart enzyme of another species as compared to the muscle enzyme of the same species. This led to the study of changes in lactate dehydrogenase during development in chickens, and it was discovered that the type of LDH that occurred in the embryonic chick breast muscles was actually the heart type. He observed that during development the genes for the M types were being expressed at an increased rate and it became the principal LDH type at the time of hatching. A connection was made to an observation that Markert had reported with regard to the fact that LDH was actually a tetramer. Their results supported the view that there were five forms of LDH consisting of the two parent types, occurring as H4 and M4, with three intermediate hybrid types, which migrated predictably in between H and M forms on polyacrylamide gels.

How did Nate make all this work so well? He somehow led a large and diverse research group that studied the biochemistry of DPN (not NAD) and many other subjects. While he was at Brandeis, he had brought on a key laboratory staff member from Holland, Johannes Everse, who worked with him for 16 years.

Part III. The move to University of California, San Diego

Nate Kaplan came to the new University of California, San Diego in 1968 at the urging of Martin Kamen, with W.D. McElroy as Chancellor. “Nate’s laboratory made important contributions in biochemical research at UCSD. Using NMR, his students and postdoctoral fellows established the conformations of the pyridine nucleotide coenzymes and other nucleotides in aqueous solution. Other important contributions were on the development of matrices for affinity chromatography of enzymes, immobilization of enzymes, and immobilization of ligands for membrane receptors.” These methods of affinity chromatography have led to a revolution in separation technology.
He wrote, “students should not lose sight of the eloquence of the experiments of Warburg because it is the same eloquence which is inherent in the isolation, characterization, and manipulation of genes.”


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