Advertisement

Remembering Our Teachers

Open AccessPublished:January 05, 2001DOI:https://doi.org/10.1016/S0021-9258(18)44198-1
      The spotlight on the biochemistry stage moves rapidly, leaving a star of yesterday in the dark and virtually forgotten. A substance, a procedure, or a biochemical event named after the star is eventually renamed and what seemed an assurance of immortality is gone. This has been the fate of the Cori cycle, the Cori ester, and of Carl and Gerty Cori as well.
      In journals (and even more so in textbooks) discoveries are described in a logical pattern unlike the sequence of erratic and serendipitous events which led to them but seldom with references to the authors included. In the hope that we might profit from reflecting on how our scientific lives and work have been shaped by past stars, I want to consider on how they (among them my teachers, Carl Cori, Gerty Cori, and Severo Ochoa) affected mine.

      Carl F. Cori (1896–1984) and Gerty T. Cori (1896–1957)

      Carl and Gerty Cori (Fig. 1) were inseparable in their personal and scientific lives and so will remain in this account. Carl F. Cori and Gerty Radnitz entered the medical school of the German University of Prague in 1914 at age 18. Carl had received his earlier schooling in the academic tradition, whereas Gerty with less academic preparation needed to absorb 8 years of Latin and 5 years of mathematics, physics, and chemistry to qualify for medical school, all of which she completed in 1 year.
      Figure thumbnail gr1
      Figure 1Gerty T. Cori and Carl F. Cori (c. 1947) in her laboratory.
      Married in 1920 after their graduation and of like minds, they looked for opportunities to do science rather than clinical practice, but there were few places available to them. The turbulence after World War I, the virulence of anti-Semitism, and the uncompromising prejudice against women scientists made them seek refuge in America. There in 1922 they were offered jobs in Buffalo, New York in the New York Institute for the Study of Malignant Diseases, since renamed the Roswell Park Memorial Institute. With little equipment and few supplies they developed quantitative and precise methods to discover connections among what seemed then to be unconnected metabolic events: glucose converted to lactic acid during muscle contraction and stored as glycogen until needed again. The cycling of lactic acid and glucose between liver glycogen and muscle operated under the influence of hormones, including epinephrine and the newly discovered insulin. The Cori cycle (Fig. 2) was never as celebrated as was the circulation of blood discovered by William Harvey three centuries earlier. Nor was the Cori cycle defined molecularly as was the citric acid cycle postulated two decades later by Hans Krebs. Yet, the Cori cycle offered deep insights into the use and storage of energy and had an indelible influence on the understanding of the hormonal control of bioenergetics.
      Figure thumbnail gr2
      Figure 2The Cori cycle. How glucose for muscle contraction is supplied from its storage as glycogen in liver and muscle.
      Well known by 1931 and having published many papers together with Gerty on the Cori cycle, it was Carl who was offered the Professorship of Pharmacology at Washington University School of Medicine in St. Louis, whereas Gerty would remain a Research Associate there. Despite the discouragements to women in science and the lack of facilities and equipment, they took off in a totally new direction.
      No longer satisfied with describing the transit of glucose in the intact animal, they began to examine its fate in vitro. In water extracts of finely minced frog muscle, they found that the glucose was converted to something novel that they identified as a new sugar phosphate ester, glucose 1-phosphate, soon called the Cori ester. (One of my ambitions for fame upon entering biochemistry in 1946 was to discover a new ester. I would then be immortalized as had Arthur Harden and William Young with fructose 1,6-diphosphate, Carl Neuberg with fructose 6-phosphate, Robert Robison and Gustav Embden with glucose 6-phosphate, and more recently, Isidor Greenwald with 2,3-diphosphoglycerate.)
      How was the Cori ester produced and what was its fate? These questions led to the enzyme studies that became the central focus of the Cori laboratory for the next decades. The most remarkable discovery was glycogen phosphorylase, which in 1938–1939 startled the biochemical world, because the enzyme not only used phosphate to split (phosphorylyze) glucose from the ends of chains to produce glucose 1-P but in reverse could elongate a glycogen polymer by serial additions of glucose from glucose 1-phosphate. This was the first large biological molecule made outside a living cell. Also, their analysis of alternative structures of glycogen phosphorylase led them to the discovery that an enzyme function can be regulated by phosphorylation, dephosphorylation, and an allosteric effector.
      After phosphorylase, many glycolytic enzymes were purified by the Coris and others who came to their laboratory. With the expert guidance of Arda Green, an outstanding protein chemist who joined them in 1942, some of these purified proteins were crystallized, including phosphorylase. By 1947, when after a year in Severo Ochoa's laboratory, I came to St. Louis (as a commissioned United States Public Health Service officer on a 6-month assignment), the Cori laboratory was the mecca of enzymology. During and immediately after World War II following destruction of European laboratories, the Coris were at the forefront of biochemistry, focused on enzymes. They welcomed refugees from everywhere to join them. Unlike the prevailing culture in American academia, they showed no discrimination toward men or women, husbands with scientist wives, Jews, or gentiles.
      The Coris, who became my most devoted patrons, were generous to accept the novice in science I still was in 1947. Although I had finished college in 1937, 3 years ahead of schedule, I was now only a sophomore in biochemistry at what seemed a rather advanced age. I felt that I had squandered nearly 10 years in clinical training and studies of rat nutrition. In daring to choose to be a biochemist, I was reassured by the Coris whose career path from clinical medicine to enzymology had been even more extended and who often asserted that medical training was useful because it widened one's view of bioscience.
      The Cori laboratory in 1947 was the most vibrant place in biochemistry. Scientists from everywhere flocked there to share in the excitement at the frontiers of intermediary metabolism, bioenergetics, enzymology, and protein chemistry. When I arrived, I was audacious enough to seek the answer to aerobic phosphorylation. That failed, but working with Olov Lindberg, a postdoctoral fellow from Stockholm, I did discover a seemingly dull enzyme, nucleotide pyrophosphatase, which eventually led me to coenzyme biosynthesis and beyond. It was in the Cori laboratory that I first learned from J.-M. Wiame, a postdoctoral fellow from Belgium, that the diagnostic metachromatic granules in Corynebacterium diphtheriae that I had observed as a student in medical school consisted of inorganic polyphosphate, the polymer of my current infatuation.
      A dramatic event that spring was the imminent visit of Hugo Theorell, the celebrated Swedish biochemist who would win the Nobel Prize in 1955. The gala banquet planned by the Coris for the evening of his arrival included the social, artistic, and scientific aristocracy of St. Louis. Gerty acquired a hairdo, used make-up, and bought a new dress for the first time in anyone's memory. Olov Lindberg went to the airport to meet the flight, but Theorell was not on it. When he came back to the laboratory without the guest, Carl asked for the first time to examine the telegraphic correspondence. Sure enough, Theorell was due exactly a week later. Poor Olov was devastated and when Theorell did appear, the fever had abated.
      The Coris always exuded a contagious work ethic, optimism, and a broad view of bioscience. After Carl and Gerty, six more would receive Nobel Prizes based on the training and outlook received at that time from the Coris and the ambience around them: Severo Ochoa and myself (1959), Luis Leloir (1970), Earl Sutherland (1971), Christian de Duve (1974), and Edwin Krebs (1991).
      Carl and Gerty complemented each other in every way. Gerty would flit through the open door to Carl's tiny office throughout the day with results from the laboratory in which she was totally engaged. Carl was calm and analytical in contrast to Gerty, who was agitated and intuitive. They gossiped about people and events—Carl with amused concern, Gerty with intensity and compassion. I recall Gerty waving a newly arrived journal, “We've been attacked”; on careful search of the paper for the offensive slight, I could not find it.
      Carl's erudition and formidable intelligence could be intimidating. He could also be dismissive. To a request from Christian de Duve in 1947 to spend 6 months in his laboratory to pursue an important idea about insulin action, Carl responded that he did not accept people for less than a year and, “With you,” he added, “there is the additional difficulty that we do not see eye to eye with respect to the action of insulin.”
      Upon returning to the National Institutes of Health from St. Louis, I was asked to form an Enzyme Section, which I could do by inviting Leon Heppel and Bernard Horecker to join me. Together with Herbert Tabor, the four of us held daily lunch seminars in which we digested virtually every paper in this Journal even more thoroughly than our brown bag lunches. Once when Gerty Cori visited me on the occasion of a National Science Foundation board meeting, she lamented my being trapped in a government laboratory. I tried to reassure her that with full freedom and resources to do what I wished and with bright and motivated colleagues, I was enjoying an ideal academic environment.
      The Coris had been largely responsible for my being offered the chairmanship of a newly created Microbiology Department at the Washington University Medical School in 1953. It had been a difficult transition for me to move from the National Institutes of Health. Among the problems were the brutally hot summers in St. Louis, from which I managed to escape to California. When I had to tell the Coris in 1957 that I decided to move to Stanford (taking the St. Louis claim to be “The Gateway to the West” literally), Carl was for the first time angry with me and sputtered: “Where will you go on vacation?” To which Gerty, close by, said: “Carlie, maybe we should have gone to California when they asked us.”
      In 1947, the Coris could celebrate the award of the Nobel Prize for Medicine or Physiology “for their discovery of the course of the catalytic conversion of glycogen”; an equal share went to Bernardo Houssay of Argentina for his discovery of the role of the anterior pituitary hormone in sugar metabolism. That year also brought the ghastly revelation that Gerty had a fatal anemia. For the next 10 years of her life, despite frequent transfusions, pain, and fatigue, she maintained a full schedule of work, travel, and social activity. This fortitude and courage enabled her to do her pioneering work on molecular diseases and complete what I will later refer to as the Cori cycle II.
      Among my many debts to the Coris is their discovery of glycogen phosphorylase whose actions encouraged me to seek an enzyme for DNA synthesis. It would be a mistake to assume that my discovery of DNA polymerase and the mechanism of DNA replication was inspired by the epochal Watson and Crick paper, which 2 years earlier had proposed that the spontaneous assembly of nucleotides in the synthesis of a DNA chain was directed by base pairing with each strand of the parental duplex. Glycogen phosphorylase, not base pairing, was what led me to DNA polymerase.
      Having been educated by the Coris, Severo Ochoa, and other stars in the galaxy of that time, I had adopted the dogma that enzymology is the most effective way toward understanding biologic events. I had found enzymes that assemble the coenzymes (NAD, NADP, FAD) and the pyrimidine and purine nucleotides, and now I hoped to go further to find the enzyme of DNA synthesis. I had in mind an enzyme, which like the Cori glycogen phosphorylase would extend a DNA chain by successive additions of a properly activated nucleotide. I never imagined that my assays would lead to an enzyme, unlike any others, that took directions from its substrate and would assemble nucleotides by Watson-Crick base pairing to create a complementary copy of the parental DNA chain.
      Gerty read widely and voraciously in history, biography, and modern novels, as well as in science. Aware of Oswald Avery's paper showing that DNA was the genetic substance, she made it a point to tell me: “You must read this. It is very important.” She had recognized the significance of DNA at least 5 years before it was given proper attention by the celebrated genetics group at the California Institute of Technology. In relating genetics to enzymes, she had been fascinated by a number of severe diseases in children characterized by excessive glycogen storage. She showed that each disease was because of a singular genetic error responsible for either a defective or missing enzyme in glycogen metabolism. The diagnoses could be made simply with assays of a tiny liver biopsy.
      Although Harvey Itano and Linus Pauling had earlier shown sickle cell anemia to be due to an inherited alteration in hemoglobin, it was Gerty's enzymology that inspired the great surge of medical investigation and diagnosis of a large number of enzyme deficiency diseases. Cori cycle II (Fig. 3) is their odyssey from clinical medicine to physiology, to enzymology and genetics, and finally full circle back to clinical medicine.
      Figure thumbnail gr3
      Figure 3The Cori cycle II. Sugar metabolism physiology in diabetes led to the enzymology of intermediary metabolism, then to the genetics of glycogen storage diseases, and finally to clinical diagnosis by enzyme assays of a liver biopsy.
      The Cori cycle and the Cori ester have been renamed, and attributions to the Coris are reasonably buried in the dustbin of history. But the lives of Carl and Gerty Cori and their monumental achievements deserve to be remembered as much as those of the political, military, arts, and sports stars of their era.

      Severo Ochoa (Se-ver′-o O-cho′-a) (1905–1993)

      Perhaps the most important feature that characterizes the few who emerge from the pack of trained, intelligent, motivated scientists is the capacity to withstand distractions and disappointments in life at home and at large, in institutional duties and politics, and in lack of resources and recognition, and also to resist the temptations of fame and fortune. Severo Ochoa (Fig. 4) was one of those few.
      Figure thumbnail gr4
      Figure 4Severo Ochoa at a Beckman model DU spectrophotometer (c. 1955).
      Born in the coastal village of Luarca in the Asturias province of northern Spain, Ochoa enrolled as a medical student at the University of Madrid in 1922. Although Ramón y Cajal, whom he idolized, had just retired, his fame still inspired those who might pursue a research career. There were few if any graduate studies in the biomedical sciences (either in Spain or elsewhere in Europe at the time), and the medical curriculum was the only recourse. Upon completing his medical courses and with no inclination toward clinical practice, he directly sought opportunities for experimental work.
      Ochoa's peregrinations from one laboratory to another (nine in Europe and three in the United States) were determined in part by seeking out those at the frontier of chemical aspects of physiology and metabolism but even more so by circumstances far beyond his control. Social and political turbulence and a state of war in Spain, Germany, and the United Kingdom drove him from one refuge to another. Buffeted by all these events, he was unwavering in his devotion to science. He always remained on course in the face of all kinds of adversities, experimental and societal. His conviction that hard work would be rewarded sustained him during the most difficult hours and permeated the atmosphere around him.
      Ochoa's very first paper was in English as was his second, a micromethod for creatine published in 1929 in this Journal, the first of hundreds that would appear in these pages over the next six decades. Upon completion of his medical thesis, he set out for Germany to spend the next 2 years in the laboratory of Otto Meyerhof (first in Berlin and then in Heidelberg). Meyerhof was renowned for his work on the energetics of muscle contraction for which he had been awarded, along with A. V. Hill, the Nobel Prize in Physiology or Medicine for 1922. To quote Ochoa: “Meyerhof was the teacher who most contributed toward my formation and the most influential in directing my life's work.”
      In 1932, with an appreciation of the importance of cell-free systems, Ochoa worked on his first enzyme, glyoxylase, with H. W. Dudley at the National Institute of Medical Research in London. Returning to Madrid, he worked on the chemistry of muscle contraction, but with the outbreak of the Spanish Civil War in 1936, he once again sought haven for a year in Meyerhof's laboratory in Heidelberg. This time he explored the action of cozymase, later known as DPN (diphosphopyridine nucleotide) and currently as NAD (nicotinamide adenine dinucleotide).
      Meyerhof, with his status under attack by German racial laws, wrote A. V. Hill in the United Kingdom who found a place for Ochoa for a year in the Marine Biological Laboratory in Plymouth. After that he was able to join Rudolph A. Peters in the biochemistry laboratory of Oxford University where, in studies of thiamin pyrophosphate in pyruvate metabolism, he was lured by oxidative (aerobic) phosphorylation. The obligate coupling of phosphorylation to the oxidation of pyruvic acid was observed by Ochoa and at the same time by Herman Kalckar in Copenhagen and by Vladimir A. Belitzer in the Soviet Union; Ochoa later estimated the number of phosphates fixed per oxygen atom consumed (P:O ratio) to be near three. Once again, the state of war in the United Kingdom enveloped all research activity and drove Ochoa, an alien, to accept in 1941 an invitation to St. Louis to join the laboratory of Carl and Gerty Cori.
      In explorations of phosphorylation in disrupted liver tissue, he found a curiosity, inorganic pyrophosphate (PPi). Because its source and fate were so vague, the finding was never published. During the year (1946) that I spent in Ochoa's laboratory, he mentioned the PPi phenomenon a number of times to Efraim Racker, Alan Mehler, and me at lunch in the cafeteria we called “Salmonella Hall.” However, its strangeness made it difficult to retain the experimental details of its origin, and we would pester him to repeat the story. Even Ochoa's patience could be tried, and finally he forbade any further mention of PPi. When I joined the Cori laboratory the next year, I tried to find the source of PPi. I failed then but did discover the source of PPi several years later when it emerged as the entity released from nucleoside triphosphates in the synthesis of coenzymes, nucleic acids, and also proteins, fatty acids, and key metabolic intermediates.
      After 1 year in St. Louis, Ochoa was offered a position as Research Associate in the Department of Medicine in the New York University School of Medicine, where he would remain for 32 years until retirement in 1974. With the least favorable facilities, Ochoa embarked on the discovery and characterization of the enzymes that he hoped would explain how cells derive virtually all their chemical energy. His confidence was based on the success in earlier decades in resolving and reconstituting alcoholic fermentation and glycolysis. The expectation was that discrete, isolatable enzymes would be identified as responsible for aerobic phosphorylation. The tricarboxylic acid cycle had just been proposed by Hans Krebs to explain how pyruvate was metabolized to carbon dioxide and water. Key intermediates in the cycle were the tricarboxylic acids (citrate and isocitrate), the enzymology of which Ochoa believed would help clarify aerobic phosphorylation.
      The pursuit of aerobic phosphorylation, the Holy Grail of biochemistry, inspired me to seek out Ochoa's laboratory in 1946. I wanted to learn the enzymology and the new biochemistry I had not been taught in medical school 8 years earlier. My training in internal medicine had been interrupted in 1942 when (as a commissioned officer in the United States Public Health Service) I served briefly as a ship's doctor in the Navy and then was assigned to study rat nutrition at the National Institute (sic) of Health. With the war concluded and with nutritional science in its twilight and bored with the feeding and bleeding of rats for 3 years, I was able to persuade the Director of the National Institutes of Health to let me spend some months away learning about the new and exciting world of enzymes.
      I was very fortunate that Ochoa was willing to take me, a complete novice in all aspects of biochemistry. In 1946, he occupied borrowed space in an old laboratory of Professor Isidor Greenwald in the New York University Department of Biochemistry. (Earlier, he had been summarily evicted from space in the Psychiatry Department in the Bellevue Hospital of the Medical School; upon returning from a concert one Sunday afternoon, he found his desk and equipment moved out into the hall.) When I arrived, his group consisted of a graduate student (Alan Mehler) and two technical assistants. Initially appointed as a Research Associate in Medicine and in 1945 as an assistant professor at the advanced age of 40, his stature in science was recognized the next year with the offer of a full professorship and chairmanship of the Department of Pharmacology. His reluctance to accept this promotion and associated responsibilities was characteristic of his indifference to academic titles and authority. “Why do I need a professorship?” he asked Efraim Racker, close by in the Department of Bacteriology. “I can do my work where I am now. Will the research work not suffer if I become a department chairman?” What finally persuaded him were two modern, well equipped laboratories developed by the previous chairman, James A. Shannon, later the Director of the National Institutes of Health. It was only in 1954 that Ochoa moved back across First Avenue to assume the vacated chairmanship of the Department of Biochemistry.
      A prized possession of Ochoa's then was one of the new and scarce Beckman DU spectrophotometers (Fig.4) (valued then at $1,500) and granted to him on loan, as was the policy of the American Philosophical Society. Patterned along the lines of the instrument devised by Otto Warburg in Germany, it was the highly effective successor to the laborious and insensitive respirometric assays of metabolic reactions that had been relied upon for several decades.
      With the spectrophotometer in constant use, we were all in dread that “the philosophical Beckman,” as it was called, would be reclaimed, but it never was. It remained and died of old age. Those early months in 1946, learning the rudiments of dynamic biochemistry, enzyme fractionation, and spectrophotometry, were the most exciting in my life. I was awed by enzymes and fell instantly in love with them. I have since had love affairs with many enzymes (none as enduring as with DNA polymerase), but I have never met a dull or disappointing one.
      The day I came into the Ochoa laboratory after Christmas in 1945 several pig hearts fresh from the slaughterhouse awaited me. My project was to separate aconitase (an activity that converts citrate in two stages to isocitrate) into its two presumed component enzymes. Starting with a water extract of the ground-up heart muscle, I tried over the next months, using ammonium sulfate fractionation and other maneuvers, but failed to separate aconitase into two discrete enzymes. (Perhaps this explains why the Japanese translation of my autobiography,For the Love of Enzymes, is entitled It Started with a Failure.) Some years later when aconitase was purified to homogeneity by others, it was found to be a single polypeptide.
      As events proved, the separated enzymes of the citric acid cycle could account for the oxidative reactions but not for the bulk of the energy captured by aerobic phosphorylation. Unlike the energetic couplings in alcohol fermentation and glycolysis, intact mitochondria were later discovered by others to be the means used in generating a proton motive force that results in the ultimate coupling of ATP synthesis to oxidative steps in the cycle.
      A chance discovery in 1955 proved to be the cited basis of Ochoa's Nobel Prize Award 4 years later. Marianne Grunberg-Manago, a postdoctoral fellow, while exploring possible mechanisms of aerobic phosphorylation, observed an activity in a bacterial extract that exchanged [32P]Pi into ADP. The responsible enzyme, purified with Ochoa's urging and direction, astonishingly converted ADP and other nucleoside diphosphates into RNA-like (NMP) n polymers.
      nADP[AMP]n+nPi
      Equation 1


      [P32]Pi+[AMP]n[AMP]n1+[P32]ADP
      Equation 2


      The initial hope that this enzyme, named polynucleotide phosphorylase, might be responsible for the biosynthesis of ribonucleic acid (RNA) was dispelled by the lack of a requirement for a DNA template to direct the assembly of a specific RNA message, the indiscriminate assembly of a polymer of any one or a mixture of nucleoside diphosphates, and finally the discovery of true RNA polymerases, which copy DNA templates with great specificity using nucleoside triphosphates rather than diphosphates. The role of polynucleotide phosphorylase in the bacteria in which it has been found is the disposal of RNA with salvage of its precious nucleotides.
      Although polynucleotide phosphorylase was disappointing for its lack of a biosynthetic role, it was the first enzyme that could make RNA-like chains and proved to be of great value in deciphering the genetic code. After Marshall Nirenberg's discovery that polyuridylic acid, (U–U–U) n , can encode a protein-like polymer of phenylalanine, Ochoa (with help from Leon A. Heppel) employed polynucleotide phosphorylase to synthesize a variety of RNA-like polymers, which were then used to identify many of the nucleotide triplets that encode the amino acids in the synthesis of proteins.
      The 1959 award of the Nobel Prize in Physiology or Medicine to Ochoa and me “for their discovery of the mechanisms in the biological synthesis of ribonucleic acid and deoxyribonucleic acid” could have substituted “RNA-like polymers” for “ribonucleic acid” in anticipation of the role these polymers would play in the elucidation of the genetic code. Ochoa could reasonably have shared the 1968 Nobel Prize with R. W. Holley, H. G. Khorana, and M. Nirenberg, cited “for their interpretation of the genetic code and its function in protein synthesis.”
      After the Nobel Prize ceremonies in Stockholm in 1959, a hero's welcome would have awaited Ochoa in Spain, but his ties to the defeated Republican government and opposition to the Franco regime made return impossible for him at that time. He had become a United States citizen, a devoted New Yorker, and a true internationalist in spirit. Nevertheless, he persuaded me, despite my own strong aversion to Franco, to go to Spain in his stead and arranged an attractive itinerary with receptions by his intimate friends and relatives. My late wife, Sylvy, and our three sons (Ken, 9; Tom, 11; and Roger, 12) were given the most affectionate welcome and expression of kinship in our opposition to their fascist government.
      To illustrate the remarkable influence of Ochoa's enthusiastic and optimistic personality, I will relate a vignette engraved in my memory. It took place in December 1946. Nearing the end of the year in his laboratory, my most formative year in science, I was about to leave for the Cori laboratory in St. Louis. With Mehler, we had discovered and partially purified the malic enzyme that catalyzed the reaction: malate ⇌ pyruvate + CO2.
      Now we were completing a very large scale preparation starting with several hundred pigeon livers. Four of us including Morton Schneider, Ochoa's talented and devoted assistant, had worked for several weeks to reach the last step in which successive additions of alcohol finally yielded the precipitate which we believed, from small scale trials, would have the enzyme in an adequate state of purity. We had only to fill in some details in a paper we had already prepared for publication.
      Late one night, Ochoa and I were dissolving the final enzyme fraction, which had been collected in many glass centrifuge bottles. I had just poured the dissolved contents of the last bottle into a measuring cylinder that contained the entire enzyme fraction. Then I brushed against and overturned one of the empty, wobbly bottles on the crowded bench. That bottle knocked over another and the domino effect reached the cylinder with the enzyme. It fell over and all of the precious material spilled on the floor. It was gone forever. Ochoa tried to be reassuring, but I remained terribly upset. By the time I got home by subway train an hour later, Ochoa had called several times because he was so worried about my safety.
      The next morning back in the laboratory I glanced at the supernatant fluid beyond the last fraction. I might have discarded it because in our trial procedures it had been inactive. However, I had saved and stored it in the freezer at −15 °C and now noticed that the previously clear fluid had become turbid. I collected the solid material, dissolved it, and assayed it for activity. “Holy Toledo,” I shrieked. This fraction had the bulk of the enzyme activity and was severalfold purer than the best of our previous preparations. Severo came running over to share my relief and pleasure, greatly amused by the “Holy Toledo.”
      Why did I save and assay the fraction we assumed was inactive? Because Ochoa's enthusiasm and optimism was infectious. Rather than suffusing a blinding intelligence, Ochoa taught me that with an ethic of unremitting experimental work, good things eventually happen. I believed they would for me as they had for him.
      Fascinated by every aspect of biochemistry and involved in all, his work ranged from muscle contraction and photosynthesis to vitamins and virus replication. He delved into the intricacies of the synthesis and breakdown of carbohydrates, lipids, nucleic acids, and proteins and played a major role in the drama of the genetic code. A courtly, charming, El Greco-like figure, intensely competitive and ambitious, he was eager to describe his latest findings, absorb those of others, and at times even appeared to intrude in all domains with little concern.
      To celebrate his 70th birthday in 1975, Ochoa chose as guests the scientists he most respected worldwide. Symposia and celebratory dinners, starting in Barcelona, were followed by a visit with Salvador Dali in his museum in his hometown in Figueras and culminated in a gala of events in Madrid. It was a party, the likes of which has not been seen in scientific circles before or since.
      Throughout his career, Severo had the constant and loyal support of his wife, Carmen. While in New York, they were the most gracious hosts in their modest apartment to an uninterrupted parade of students, postdoctoral fellows, visiting scientists, and colleagues. They especially enjoyed music, fine food, travel, and good company. With no children and attachments to his beloved New York weakened by the loss of most of his contemporaries in science, he and Carmen finally returned to Madrid in 1985. Her death shortly thereafter was a loss from which he never recovered despite the adoration of devoted family, friends, and students.
      To the legion of postdoctoral fellows, students, and sabbatical guests who came to him from every corner of the world and left to become leaders in science and to the Spanish nation, Severo Ochoa will live on in their memory as a great teacher and an inspiration for the pursuit of science.

      Envoi

      In this brief essay, I have regretfully not included H. A. Barker, Herman Kalckar, Efraim Racker, Harland Wood, and so many others of my colleagues and students who have also been my teachers.

      BIBLIOGRAPHY

      Carl and Gerty Cori

      McGrayne, S. B. (1993) Nobel Prize Women in Science, Birch Lane Press, Carol Publishing Co., New York
      Cori, C. F. (1969) The call of science. Annu. Rev. Biochem. 38,1–20
      Kalckar, H. M. (1958) Gerty T. Cori. Science 126, 16
      Ochoa, S. (1958) Gerty T. Cori, biochemist. Science 126, 16
      Randle, P. (1986) Carl Ferdinand Cori. Biogr. Mem. Fellows R. Soc. 32, 67
      Cohn, M. (1992) Carl Ferdinand Cori. Biogr. Mem. Natl. Acad. Sci. 61,78–109
      Larner, J. (1992) Gerty Theresa Cori. Biogr. Mem. Natl. Acad. Sci. 61,110–135

      Severo Ochoa

      Kornberg, A., Horecker, B. L., Cornudella, L., and Oró, J. (eds) (1975)Reflections on Biochemistry. In Honour of  Severo Ochoa.Essays by participants in the celebration of his 70th birthday. Pergamon Press, New York
      Kornberg, A. (1993) Severo Ochoa. Nature 366, 408 (obituary)
      Kornberg, A. (1997) Severo Ochoa. Proc. Am. Philos. Soc. 141, 479–491
      Losada, M. (1994) Ochoa.Hombre de Ciencia y de Bien. Secretario de Publicaciones de la Universidad de Sevilla, pp. 1–48 (in Spanish)
      New York Times (1993) Severo Ochoa, biochemist, a Nobel winner 88, dies. November 3. Obituary
      Ochoa, S., and Valdecasas, J. G. (1929) A micromethod for the estimation of creatine in muscle. J. Biol. Chem. 81, 351
      Ochoa, S. (1980) The pursuit of a hobby . Annu. Rev. Biochem. 49, 1–30
      Sols, A., and Estrevez, C. (eds) (1975) Trabajos Reunidos de Severo Ochoa 1928–1975.Published by Servicio de  Publicaciones, Ministerio de Educacion y Ciencia Cuidad Universitaria, Madrid-3, Spain. Reviewed by A. Kornberg  in Trends Biochem. Sci. (1976) 1,267–268