Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Author: Larry H. Bernstein, MD, FACP

Nathan Oram Kaplan

Continuation of a portrait

http://pharmaceuticalintelligence.com/2013/01/26/portrait-of-a-great-scientist-and-mentor-nathan-oram-kaplan/

Notable Investigations

When Alan Wilson joined Nate’s laboratory, perticularly interested in molecular evolution they carried out studies that revealed many interesting taxonomic and enzymatic relationships. For example, the flatfish, halibut, flounder, and sole have only the anaerobic type of LDH(M4) in their tissues as adults. Since they bury themselves in the sand and live in an anaerobic environment, one can see why the M-type enzyme is present in the heart of these fish. When the flatfish larvae hatch they are free-living forms. They have one eye on each side, but at the time when one of the eyes move from one side to the other there is also a change in the lactic acid dehydrogenase. The young fish have the H type present in heart and other tissues and at the time of the eye movement there is a change to the M form. When Nate and Alan examined a number of invertebrates, they found that the arthropods, particularly the lobsters and crabs, had a very unusual LDH.

Margaret Ciotti worked with Kaplan on the horseshoe Crab (Limmulus). The rate of enzymatic lactate oxidation with NAD was very slow, but this group of arthropods had high activity with the acetylpyridine analog of NAD (AP-DPN). To their dismay that this LDH reacted very well with the natural coenzyme in contrast to other crabs. It was simple! Limmulus isn’t a crab. An examination of spiders, scorpions, and tarantulas revealed that all had enzymes similar to horseshoe crab. It soon became apparent to Nate that the horseshoe crab was related to the spider species and not to the lobster-crab group. It was very surprising to Nate that he would be able to classify an organism using enzymological techniques which agreed with the taxonomic classification. In addition, the Limmulus enzyme would not work with pure L-lactate. Pure L-lactate was not a good substrate but D-lactate was very effective because the LDH for Limmulus was D-LDH. This was surprising, because almost all animal LDH’s are of the L-configuration and are tetrameric.

A major publication about the structural differences between the H4 and M4 isoenzymes and the active site was published in PNAS 1974. Eventoff et al.
Proc. Natl. Acad. Sci. USA 1977; 74(7):2677-2681 [Biochemistry]
The changes in the hydrophobic adenosine binding pocket [residues 96(94), 119(116), and 55(54)] tend to reduce the size of the amino acid side chains in the H4 isozyme, increasing the volume within the pocket. Thus, in the initial binding of the cofactor (38), the adenosine will bind with reduced affinity to the H4, as compared to the M4, molecule. One substantial difference is the presence of Gln 31 in the heart enzyme and Ala in the muscle enzyme. The glutamine could form a hydrogen bond with the nicotinamide phosphate (thus increasing the energy of binding for the heart-isozyme) and move the nicotinamide end of the coenzyme out of the active center by at least 1 A. Unfortunately, the evidence for this important change is not clear in the electron density maps. The relative position of the substrate and the nicotinamide would favor the formation of the NAD-pyruvate covalent adduct between the methyl group of pyruvate and carbon 4 of nicotinamide in the H4 isozyme. Thus, the inhibition by pyruvate due to the formation of the abortive ternary complex LDH:NAD-pyruvate can occur more easily.

Nate received a telephone call from the Bureau of Fisheries in Boston inquiring if they could distinguish between haddock and cod using their techniques. There was a suspicion that the filets being sold in the market were not haddock but mostly cod. Haddock sold at four times the cost of cod. Most of the fillets contained almost exclusively cod muscle LDH. Today all the filets of haddock are tested for the presence of cod. These observations were put into the Congressional Record as an indication and illustration of how molecular biology could be of value to the average consumer.

A fresh endeavor

In the 1970s, he and Gordon Sato (previously at Brandeis) established a successful colony of athymic mice. Since these mice lacked a thymus, human tumors were not rejected and tumor xenografts could be grown in the animals.The mice were used to examine anti-cancer agents making this facility an important component of the UCSD Cancer Center“.

What transcends his scientific accomplishments was the warm and inspiring influence that Nate Kaplan had on those who worked with him. Young investigators from the world over were drawn to his laboratory, where they were accommodated with excellent research problems, excellent facilities, and the qualities of the man himself. These qualities were a combination of warmth, understanding, keen insight, and a contagious enthusiasm for biochemistry which permeated all of his professional activities.

Correspondence with Johannes Everse, Emeritus Professor of Biochemistry, Texas Tech Univiversity, Lubbock, TX

What strikes you about the unique character of Nathan Kaplan?

I would like to emphasize one more of Nate’s qualities: not only did he have an extraordinary keen ability to recognize good people, once you were part of his group, he did go out of his way to help anyone who was in trouble or who wanted do more than he expected of them. I believe in Yiddish they would call him a MENSCH. I worked with Nate for 16 years, from 1960 till 1976, and I owe him my career.

One of the best examples of his ability to pick up good people was Francis Stolzenbach. Fran came to Nate’s lab in Baltimore as a high school graduate with nothing to recommend him. Nate needed someone to do some routine laboratory chores. After a short time Nate realized that Fran was not only smart, he also learned fast and could work accurately, but he could always count on Francis that the work got done. Francis got himself involved in a nasty divorce, had a drinking problem, and a few other things like that, but Nate was always ready to help him out of his problems. As you know, when Nate moved to Brandeis, he asked Francis to go with him and later the same when he moved to San Diego. You know that Nate trusted Francis with keeping the research going, even when Nate went to Israel for a few months. Without any formal education, Francis became an excellent researcher who knew how to move a project forward and was capable of supervising even postdoctoral fellows when asked to do so. I give Nate credit for recognizing that Francis had the ability to do that not long after Francis joined him in Baltimore.

Let me also tell you what Nate did for me while I was in his laboratory. I started my career as a laboratory technician. After high school I first worked for 3 years in the lab of a factory like Del Monte, where they taught me how to do quality control. That ended when I was drafted for the Army, which lasted only one year. After that, I found a job at a pharmaceutical factory near Amsterdam, where I worked on a procedure to synthesize Vitamin A for four years, while I earned my technician certificate in organic chemistry. After that I moved to the biochemistry laboratory, where I worked for another 4 years earning my certificate in biochemistry. At that point I was 28 years old and had climbed the corporate ladder as far as possible; the next step up required a university degree, which I did not have and could not get. The Company had a very nice policy: they hired professors from the University of Amsterdam and Utrecht to come and give courses in the evenings for employees that were interested. After completing the course and taking an exam, one got a certificate of completion. There were lots of different courses on different scientific topics. I took a number of these courses during those 8 years.

But I felt unhappy that at the age of 28 I had no way to advance my career further. At that time (1959) the company research labs were always looking forward to the latest issues of Science and Nature and other scientific journals in order to pick up the latest methodology and findings. The USA had the reputation of being the best country in the world for research. So I talked with my supervisor about me taking a leave of absence and go spend a few years in the States to learn how they do research. He thought I was crazy, but after some more talking he agreed. So I started applying for any position that was advertised in Science that seemed suitable for an experienced laboratory technician. After trying for about 6 months, I got a phone call from the secretary of a professor in Amsterdam, who wanted to know if I would like to be interviewed by the professor for a job that a friend had available in the USA. I went and about a month later I got a letter from Dr. Lowenstein at Brandeis University, telling me that they were offering me a job. I had never heard of either Dr. Lowenstein or Brandeis, but I knew that a job at a University would be far preferable to a job at some company. So I accepted, figuring that if this was not the right job, I could always look for something better once I was in the States. In the meantime, Brandeis had started the procedure to get me a first preference visum, and one day I got a big envelope with lots of papers that I had to take to the US Consulate in Rotterdam. It was the visum application, but that application was signed by Nathan O. Kaplan. I knew that name! So I arrived at Brandeis in June 1960, while Nate was in Israel for the summer. When he came back, he introduced me to Dr. Lenny Brand, then a postdoc, to be his assistant. A year later, when Lenny went to Johns Hopkins, I became Kaplan’s assistant. By that time I had forgotten about going back to Holland. I felt like I had joined one of the best research groups in the world.

Then in 1963 I decided one day to listen in on the biochemistry course that was given to the first year students. I discovered that they were using the same biochemistry textbook that I had used in Holland for my technician certificate. I figured that if the graduate students start at that level, then I should also be able to do these studies. So I took a tranquilizer to calm my nerves, went to Nate’s office, and asked him if there was any possibility for me to earn a university degree. I expected that he would smile at me and tell me to get back to work, but instead he told me to sit down and started to ask me lots of questions about my background. Remember, I came to the US with nothing more than a Dutch high school diploma, two technician certificates and a bunch of certificates that I had taken various courses, all things that nobody could read. I of course realized earlier that I was invited to come to Brandeis almost solely on the recommendation of that prof in Amsterdam and my own explanation of what the requirements were to earn the technician certificates. It was simply: OK, you are here now, let’s see what you can do!

After Nate questioned me for about an hour, he told me to my surprise that he would talk to the Dean! A few days later he told me that the Dean wanted to be assured that I could do academic work, and wanted me to take an undergraduate course of my choice. I chose to take math 101, because I knew nothing about differentials and integrals. The instructor was told that I was a “special” unregistered student, but I was to take all the assignments and exams as a regular students and graded accordingly. I got an A in the course. Then the Dean agreed to accept me as a “special” graduate student, while Nate agreed that I could take time off to take the required courses, one at a time, and make up the time during evenings and weekends. My point is that it must have taken an awful lot of persuasion by Nate to get the Dean to agree to admit someone to graduate school without ever being in an undergraduate school and with mostly papers that nobody could read or verify, just taking Nate’s word for it. Brandeis is not known as a school that is generous in handing out degrees.
Eventually, when Nate moved to San Diego, I wanted to continue working for my degree at UCSD. But UCSD, being a state school, had strict rules about requiring an undergraduate degree in order to get into graduate school. Nate moved heaven and earth trying to overcome this obstacle, but no success. About a year later, when Drs. Lawrence Levine and Bill Jencks from Brandeis came to visit UCSD and I told them about my predicament, they went back to Brandeis and convinced the department to recommend to the Dean to grant me a Masters degree. I got that in the mail in 1971. In 1973 I got my Ph.D. at UCSD. My dissertation subject: NAD adducts and ternary complexes.

Granted, Nate went out of his way for me. I often thought that he sort of had adopted me as a son or something. But during my 16 years with him I have seen him help others in a similar way when he thought they deserved a helping hand. More than anybody I have ever met, this guy had a heart of gold.

How did Methods in Enzymology come about?

Nate and Sidney Colowick learned to know each other when they both were postdoctoral fellows at the McCollum Pratt Institute at Johns Hopkins. Other people that worked there told me that Sidney was the experimentalist, whereas Nate spend more time figuring out how to approach a scientific question. In the process, Nate figured out that the most important asset in a laboratory is to have ready access to methods and procedures. At that time these were published in scientific journals, but making copies was expensive and the copies lasted only for a few months. So it was hard to remember where who had published a method for this or a procedure for that. To overcome this problem, Nate started keeping a collection of all the methods and procedures that he found in the literature and that he thought may be useful some day. Soon he had a sizeable collection, and people learned about that. So he was constantly asked “Nate, do you know how to do this? or Nate, do you have a procedure for that? It was Bill McElroy, at that time the director of the Institute, who suggested to Nate that this constant demand for the latest procedures and methods could be overcome by putting his collection into a book, so everybody would have them available. McElroy knew someone at Academic Press and that resulted in the birth of Methods in Enzymology. The initial issue consisted of 4 volumes and a few years later the series was updated with newer methods in Volumes 5 and 6. By that time Nate had found out how much time is involved in publishing a book, which left him not much time for research. Also, at that time he had been approached by Brandeis to take the chairmanship of a new biochemistry department, which would be the third such department in the US. So the series was completed with Volume 7, which was just an index of the 6 existing volumes.

But the series was extremely successful. For example, at that time I worked in the research laboratories of a pharmaceutical company in Holland, and we had the complete series in our library, where they were heavily used! Since science moves forward, Nate got constantly called upon to further update the series. But at Brandeis he could not afford the time any more. Then someone, I believe it was Willis Wood, suggested to Nate to let others edit additional volumes, while he and Sidney would become Editors-in-Chief. As such, they would still have to approve any plans for new volumes as well as approve the final product, but others would do all the “footwork” of getting the volumes together. Nate and Sidney thought that it was a great idea and, as they say, the rest is history.

There is no question that the Methods in Enzymology series has been by far the most successful series of books ever published in biochemistry and possibly in other sciences as well. It was Nate’s insight that accomplished that feat by realizing that the “rate-limiting step” in research at that time was the lack of readily available methods and procedures.

There was a lot of work on the adducts. Please comment on that.

Concerning your follow-up: The story of the NAD-adducts indeed is an interesting one, and I had a lot to do with it.

It started back in the 1950’s when someone in Nate’s laboratory made an adduct of NAD with, I believe, acetone or acetaldehyde. But except for trying to learn about the chemical properties of NAD, nothing was done with it. Then in the early 1960’s, shortly after I joined Nate’s group, a technician found that when she kept a solution of NADH in the freezer overnight, the NADH had lost most of its activity when she did her LDH assays. From that point on NADH solutions were made fresh every day. But one of the postdocs, Peter Fawcett, took a look at a frozen solution and found that it still had its original UV spectrum, ruling out that the NADH was oxidized. Then he found that the frozen NADH actually was a potent inhibitor of an LDH assay when added to a normal NADH-pyruvate assay. Peter published a short note in Biochim. Biophys Acta about this LDH inhibitor formed from NADH. But it triggered Nate’s interest in terms of what actually happened to the NADH. He put me to work on trying to find out, since I originally worked as an organic chemist in Holland. I soon showed that nothing had happened to the adenine moiety, and the riboses and phosphates were also still in place and intact. Hence, the change had to be in the nicotinamide ring. The UV spectrum indicated that it still had the dihydropyridine structure of NADH, suggesting no changes to the ring itself. I then tested for the presence of an intact amide group and got a negative result. A few more tests indicated that some carbonyl compound had reacted with the amide group and the 4-position of the nicotinamide ring. The project was set aside for a time.
Over the years I often thought about the adduct project, in part because I was a bit unhappy about having worked hard on that project for about 9 months with nothing to show for it. I already had figured out that the acetone could not react with NADH; the NADH had to be oxidized to NAD first, then it could react with acetone. This would result in no changes in the UV spectrum, but of course all NADH activity would be lost. I also knew from my organic chemistry background that for acetone to react with NAD it had to be in the enol-form instead of in the keto-form. Since it is normal for these carbonyl compounds to be present in both forms, that would not be a problem. That would mean that NAD should be able to form analogous adducts with many carbonyl compounds, especially with alpha-keto-acids. For example, a solution of pyruvate consists of 95% keto-pyruvate and 5% enol-pyruvate.
At that time we had also learned about the abortive ternary complex of LDH-NAD-pyruvate as an inhibitor of the LDH activity. This complex, when prepared in a cuvette, also showed the characteristic UV absorbance at 340 nm of NADH, suggesting that the nicotinamide ring was in the dihydro-form in the complex. So I was wondering if the abortive complex actually was an enzyme-bound NAD-pyruvate adduct. So I had Estelle Zoll prepare the pyruvate adduct of NAD and then tested it as an inhibitor of LDH. It showed potent inhibition. I then asked her to prepare the adduct of NAD with oxaloacetate, which is different from pyruvate by a single –CH2- group. This adduct had no effect on LDH, but she showed it to be a potent inhibitor of malate dehydrogenase. That made us wonder if this specific inhibition indicated that the enzymes were recognizing their respective substrates when it was bound chemically to NAD; i.e., that the adducts bound to both the coenzyme binding site and the substrate binding site of the enzymes. We then made a series of NAD adducts, using substrates of enzymes that we had access to, and found that indeed each enzyme recognized its substrates in the adducts. I was jubilant and felt very happy when I showed Nate these results.
At the same time, Dr. Michael Rossmann at Purdue was trying very hard to determine the structure of dogfish muscle LDH, using x-ray crystallography, as well as the location of the coenzyme and, if possible, the substrate. So I felt like we were approaching the same problem, using two different techniques. He used the physical technique of x-ray crystallography, whereas I was using chemistry. Eventually we got together and our results were published in a PNAS article in 1973. A detailed description of the results with the adducts and ternary complex was published in Advances in Enzymology, vol. 37, also of 1973. I can mail you reprints of these articles if you wish.

Representative Early Publications

(Nathan O. Kaplan Papers. MSS 0099. UC San Diego::Mandeville Special Collections Library)
Nicotinic acid analogue of diphosphopyridine nucleotide 1958. N box 38, folder 68.
Enzyme-coenzyme-substrate complex. of pyridine nucleotide depend. dehydrogenases 1958. box 39, folder 5.
Chemical properties of 3-substitited pyridine analogues of dpn 1959. box 39, folder 11.
Enzymatic studies with analogues of diphosphopyridine nucleotide 1959. box 39, folder 12.
Molecular heterogeneity and evolution of enzymes 1960. box 39, folder 17.
Mechanism of depletion of mitochondrial pyridine nucleotides 1960. box 39, folder 24.
Measurements of enzymes in the diagnosis of acute myocardial infarction 1961. box 39, folder 28.
Pyridine nucleotide transhydrogenase VIII. Properties of the transhydrogenase reactions of an enzyme complex isolated from beef heart mitochondria 1961. box 39, folder 33.
Heterogeneity of the lactic dehydrogenases of new-born and adult rat heart as determined with enzyme analogs 1961. box 39, folder 37.
Regulatory effects of enzyme action 1961. box 39, folder 38.
Inhibition of dehydrogenase reactions by a substance formed from reduced dpn 1961. box 39, folder 40.
Nature and development of lactic dehydrogenases 1962. box 39, folder 44.
Functions of the two forms of lactic dehydrogenase in the breast muscle of birds 1963. box 39, folder 52.
Substituted nicotinamide analogues of nicotinamide adenine dinucleotide 1963. box 39, folder 56.
Alterations of tissue lactate dehydrogenase in human neoplasms 1963. box 39, folder 65.
Lactic dehydrogenases: functions of the two types 1964. box 39, folder 67.
Evolution of lactic dehydrogenases 1964. box 40, folder 1.
Lactate dehydrogenase – structure and function. 1964. box 40, folder 4.
Lactic dehydrogenase in cancer 1964. box 40, folder 5.