Moments of Discovery: My Favorite Experiments

Even before completing my undergraduate stay at Penn State University, I had decided to get a Ph.D. in biochemistry to help me get a good job in the pharmaceutical or food technology industry. However, sometime during my last year I came across a series of papers from the Biochemistry Department at Western Reserve University in Cleveland, Ohio, an institution I had not previously considered as a place to do graduate work. Those reports described the use of radioisotopes to track metabolic reactions in vivo and in vitro. Reading further, one name, Harland Wood, dominated the list of authors (Fig. 1). Soon after World War II ended, Wood had been recruited to the Western Reserve University medical school from the University of Minnesota to resurrect a virtually moribund biochemistry department. Intrigued by the new technology, I decided to explore this new approach. By 1948 when I arrived as a graduate student, Wood's carefully chosen group of younger faculty constituted one of the top departments in the country. Because of an unfortunate miscommunication, I found that I had inadvertently applied to and been accepted into the remnant of the defunct biochemistry department. Although keenly disappointed, I tried to make the best of it by teaming up with two former members of the department, Leonard Skeggs and Jack Leonard, to develop a new kind of artificial kidney. During the ensuing 2 years, I became pretty adept at surgical removal of dog kidneys and keeping the animals alive by dialysis. Recognizing my frustration during one of the biochemistry graduate courses I was obliged to take, Wood asked if I was interested in joining his department and completing my thesis research there. It was a break that changed my life. During the next 2 years, I completed my thesis research on the mechanism by which one-carbon compounds, formate, formaldehyde, and methanol, are converted to the methyl group of methionine. Although Warwick Sakami, one of the young professors in the department, was my nominal adviser, it was Wood who provided the inspiration that set the tone of my career in research. Wood's scientific exploits as a graduate student and postdoctoral fellow were legendary among members of the department. While working for his Ph.D. at Iowa State University, he made the startling discovery that heterotrophic organisms (those that live on complex carbon compounds for their source of energy and biosynthetic needs) incorporated carbon dioxide into their cellular substituents. This property, previously believed to exist only in photosynthetic plants and autotrophic microbes, was met with considerable disbelief. Characteristically, Wood was not about to let the doubts persist and he turned to isotopes to prove his point. After a brief fling with radioactive carbon-11, whose incredibly short half-life made it difficult to use, he chose the stable carbon-13 isotope to trace the metabolic utilization of carbon dioxide. Because an enriched source of carbon-13 was not readily available in the late 1930s and early 1940s, he proceeded to prepare some himself. He built a water-cooled thermal diffusion column in a five-story abandoned elevator shaft that enabled him to separate sufficient quantities of carbon-13 for his experiments. However, he also needed a way to measure the abundance of carbon-13 in the metabolic products. Getting advice from Alfred Nier, a physicist at the university, he built a mass spectrophotometer from scratch. This display of dogged self-sufficiency reflects his early experiences on the family's Iowa farm and the remarkable work ethic he and his brothers and sisters acquired from their parents and the community in which they were raised. Wood's devotion to research and to those who shared his commitment to science showed through his outwardly gruff manner. “Hanging out” with the graduate students during the many late evenings when he lingered in the laboratory before heading home were the times I treasured most. His unremitting honesty and forthrightness in the way he practiced science provided the model we all tried to emulate. Wood's response to criticism showed through during a visit from the legendary Hans Krebs. Some years before, Wood had concluded that citric acid could not be one of the intermediates in the Krebs cycle for metabolizing dicarboxylic acids. He based his conclusion on the finding of an unanticipated isotope distribution in the glucose portion of liver glycogen and in intermediates of the cycle after feeding a variety of 14C-labeled metabolic precursors. He concluded that citric acid, a chemically symmetric molecule, could not be an obligatory intermediate in the cycle. For a period, Krebs' original formulation of the cycle stood amended with cis-aconitic acid replacing citric acid. Krebs' lecture during that visit provided a wholly novel way to explain how citric acid could be metabolized in an asymmetric way, thereby accounting for Wood's findings. Alexander Ogston had pointed out that citric acid was asymmetric with respect to its chirality and that the enzyme aconitase could bind and metabolize citric acid in an asymmetric manner. Entirely free from any apparent pique, defensiveness, or embarrassment, Wood was forthright in acknowledging Krebs' ingenious explanation and admitted that in this instance the isotope result had been misleading, and he stressed the necessity of understanding the enzyme mechanisms. The magnanimity and generosity of his praise and the unself-conscious manner in which he responded brought home to me his often repeated admonition that criticism focused on the science was not meant to diminish one as a person. It was important for me to keep that in mind when he vigorously contested what I thought were clever interpretations or speculations. David A. Goldthwait and Richard W. Hanson captured the essence of the man in their National Academy of Sciences Biographical Memoir: “... as a man without pretensions, whose opinions and decisions were always based on principles and not on personal factors, a man whose mind was open to new ideas and concepts, a man who by his example and encouragement got the best out of his associates, and a man who, once he made up his mind, would drive straight toward his goal. In him one felt the warmth, strength and integrity that made him unique” (1Goldthwait D.A. Hanson R.W. Harland Goff Wood. Biogr. Mem. Natl. Acad. Sci. 1996; 69: 3-36Google Scholar). Much to the benefit of the students and faculty, Wood's reputation enticed many of the world's leading biochemists to visit and present their latest findings. Besides Krebs, the ones I recall as being most influential were Carl Cori, Severo Ochoa, Fritz Lipmann, Feodor Lynen, Albert Lehninger, Herman Kalckar, and Arthur Kornberg. The latter two made a special impression. Aside from the fact that I and most others could barely understand Kalckar's heavily Danish-inflected English, his almost childish, joyous personality made his still novel use of spectrophotometry for studying nucleotide enzymology seem all the more exciting. Arthur Kornberg was one of the rising “stars” in enzymology but equally notable for me at the time was that he and I graduated from the same Brooklyn High School, Abraham Lincoln, albeit about 10 years apart. As I was finishing the research for my thesis, I decided that it would be important for me get more intensive training in enzymology. Aware of that decision, Wood arranged for me to have a postdoctoral position with Carl and Gerty Cori at Washington University. Much to his chagrin, I told him that I preferred not to live in St. Louis because of its vestiges of racial segregation and notoriously torrid summers; perhaps apocryphal, foreign consular officials were said to receive a “tropical pay bonus” during their assignment in St. Louis. Turning down what was possibly a career-making opportunity because of an aversion to living in St. Louis was in Wood's mind shortsighted, if not foolish. Disappointing Wood in his aspirations for my future was painful, but in time my decision was forgiven, perhaps because he appreciated my independence. Having thought hard about what I would do instead, I decided to spend the first of two planned postdoctoral years working with Herman Kalckar in Copenhagen, Denmark and to spend the second year in Arthur Kornberg's laboratory at the National Institutes of Health. Herman Kalckar came to the United States in the late 1930s and was forced to remain throughout World War II. During the eight or so years he spent in the United States he was among the earliest to formulate the concept of high energy bonds as the form in which free energy was captured and stored during oxidative metabolism. Most people were captivated by his charmingly buoyant and fun-loving manner despite their inability to understand what he was saying. As I learned later, it was not just his Danish-like English that confused people, for even the Danes found him difficult to follow when he spoke his native language. Soon after the war ended and the situation in Denmark was near normal, Kalckar returned to Copenhagen and established an active laboratory at the university's Institute of Cytophysiology (Fig. 2). When I arrived in the fall of 1952, he had already assembled an interesting collection of fellows from Sweden, India, Italy, Scotland, Australia, and Canada. The international makeup of the laboratory made English the lingua franca for our scientific and social discourse. The locals in the laboratory were amused and tolerant as they listened to our futile promises to refrain from speaking English in the laboratory; their references to Danish as a “throat disease” made us feel less inadequate. James D. Watson had been in the laboratory the year before I arrived to learn some nucleic acid chemistry, ostensibly on the advice of his graduate professor, Salvador Luria. His stay, however, was brief probably because of his outspoken disdain for biochemistry and his belief that Kalckar had little interest in genes or DNA. That assessment was probably a result of Kalckar's inattention to the laboratory that year, for when I learned to translate Kalckar's mutterings, it was apparent that his interests in biology were wide ranging and most often stimulating and provocative. Although still not fully recovered from the Nazi occupation, the Danes were welcoming and extraordinarily hospitable, occasionally to our embarrassment. Living in Taarbaek, a small upscale fishing village bordering on the King's private deer park on the outskirts of Copenhagen, was a welcome relief from 4 years in Cleveland and from my wife Millie's 4 years of nursing at the university's hospital. My daily commute to and from the institute and the bike ride home through the woods to our “villa” provided the quiet time for preparing and thinking about the experiments of the day. My American Cancer Society fellowship stipend ($3600 per year), which one of our Danish friends speculated might have been more than the King's allowance, allowed us to live well and to sample the sights and culture of the Europe we had only read about. Kalckar was a dreamer, often seeking novel explanations for paradoxical observations. One of these originated from a suggestion by Thomas Rosenberg, a physicist friend from the nearby Niels Bohr Institute. Responding to a proposal that insulin acted on the phosphorylation of glucose by hexokinase, they speculated that the hexokinase reaction occurred in two steps; the first product was a high energy glucose 6-metaphosphate that was then hydrated to form glucose 6-phosphate; insulin was presumed to stimulate the hydration step. Commonly, such musings formed the basis for discussions during afternoon tea. At one discussion, I outlined a way to test that possibility. Presuming that the first step was reversible and being aware that ITP was also a substrate for hexokinase, it seemed plausible to expect a glucose-dependent transfer of phosphate from ATP to IDP or from ITP to ADP. At the time, Wolfgang (Bill) Joklik, a postdoctoral fellow from Australia via a Ph.D. at Oxford, joined the laboratory and we agreed to test that idea. Because 32P-labeled ATP and ITP were unavailable commercially at the time, we made our own. A rabbit was injected with 32P-labeled phosphate and ATP was harvested from the skeletal muscles, an exercise Bill and I still chortle over when we recall how the rabbit nearly sabotaged our effort. The experiment to test the Rosenberg-Kalckar hypothesis led to a serendipitous and more interesting result. The terminal phosphate of ATP was transferred to IDP and from ITP to ADP, but neither reaction was influenced by the presence of glucose. Clearly this finding was inconsistent with the hypothesis' predicted key role for glucose. Following up on that result, Joklik and I discovered that the transphosphorylation activity was because of a previously unknown enzyme that uses ATP to phosphorylate the four ribo- and deoxyribonucleoside diphosphates to the respective triphosphates; we dubbed the enzyme nucleoside diphosphokinase or Nudiki for short (2Berg P. Joklik W.K. Enzymatic phosphorylation of nucleoside diphosphates. J. Biol. Chem. 1954; 210: 657-672Abstract Full Text PDF PubMed Google Scholar). Although the new activity was first detected in vertebrate muscle, we purified it from yeast. Subsequently the enzyme was found to be widely distributed in pro-and eukaryotes, not surprisingly because it plays a critical role in generating the “building blocks” for RNA and DNA synthesis. One of the papers that got lots of discussion at our tea times was a report from Fritz Lipmann, Feodor Lynen, and their respective collaborators Mary Ellen Jones and Helmut Hilz (3Jones M.E. Lipmann F. Hilz H. Lynen F. On the enzymatic mechanism of A with and J. Chem. Scholar). That report that the of how organisms make had been and and the of their proposal were to my At the time, the of in was to via two separate enzymatic For and for By in and to be made by a enzyme in a The that the reaction in intermediates was provocative. 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Biol. Chem. Kornberg A. enzymatic of J. Biol. Chem. Full Text PDF PubMed Google result in the of even trace of or in their enzyme could for the despite Kornberg's I was to explore my with only the admonition to the enzyme before their and a short time, the of as the I had a purified Much to my neither of the two of reactions were about to what was needed to the I that with all the present was a of with ATP but when and were was to the to the of ATP with I that ATP with to a of with the of P. an enzymatic mechanism of Biol. Chem. the and of J. Biol. Full Text PDF PubMed Google Scholar). 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However, I was about activity in the of the one by P. the of and methionine. J. Biol. Chem. Scholar). It not to be to the enzyme discovered by that because of the of that Soon of ATP with were and their that was for a acid. The to with acid in the reaction was with the that these also the of by with the mechanism of the it seemed that was a for the a to I asked my first graduate student, to for such an our his led to the discovery of RNA as the P. enzymatic mechanism for to Natl. Acad. Sci. A. PubMed Google Scholar). of the RNA that it was small and probably to the that and had found to stimulate the in of into A acid intermediate in synthesis. J. Biol. Chem. Full Text PDF PubMed Google Scholar). work by and Jack my first postdoctoral established that a and enzyme acid to an and then the group to a RNA molecule, to as At I spent the of at his laboratory in to those in of the purified was to the to ATP with and to in the presence of P. The of J. Biol. Chem. on the enzymatic utilization of the of J. Biol. Full Text PDF PubMed Google Scholar). Because the of the could be by acid, we that acid transferred to only a set of It experiments to that had a surprisingly one of the two This acid per with that the as for to their during of On synthesis. Biol. Google Scholar). of the of and by ATP to be the first discovered of a of enzymatic DNA and are also by In the of the of the it to the joining of DNA and of the of the enzyme to In the of the to the reactions to over a of about years, were most because they at the of my career when I still many of the experiments with my they also provided the that I could do research my aversion to living in St. I learned that with remarkable in a stimulating the occasionally and and over the years of my stay the its of racial and a social and life.

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