From Oxygenase to Sleep
2008; Elsevier BV; Volume: 283; Issue: 28 Linguagem: Inglês
10.1074/jbc.x800002200
ISSN1083-351X
Autores Tópico(s)Neuroscience of respiration and sleep
ResumoI graduated from Osaka University School of Medicine in 1942 at the age of 22 and then served in the Japanese Navy as a medical officer until the end of the Second World War. On a dreary cold afternoon in the late autumn of 1945, I returned home to Osaka, where I saw that the entire city had been almost completely destroyed by air raids and that my family's house had disappeared without a trace. Under the circumstances, I decided to join my parents, who had evacuated to their rural home village of Ejiri, perhaps to help my father with his medical practice. Before leaving Osaka, I visited my former mentor, Professor Tenji Taniguchi, to tell him of my plans. He listened to me attentively and then asked me if I knew the old Japanese saying, "Take a seed of persimmon rather than a ball of rice." I was puzzled and speechless for a moment. In those days, food was scarce, and rice was rationed. A ball of white rice would be a feast! On the other hand, the seed of a persimmon would grow into a tree in 10 or 20 years and then bear plenty of flowers and fruits, which would in turn yield numerous new seeds that would grow to produce yet more trees. Standing at a crossroad of my life at the age of 25, I was faced with the following question: "Should I become a clinical doctor and practice medicine like my father, or should I become a research scientist like Professor Taniguchi?" After pondering over the Professor's remarks and discussing the situation with my father for several days, I finally decided to join Professor Taniguchi's department and started my career as a rookie microbiologist in the Department of Bacteriology, Osaka University, my alma mater. However, it was obviously not a rational decision. Believe it or not, my starting salary was only 60 yen per month, equivalent to 20 cents, not even one dollar a month! Life was miserable and so were the conditions in the laboratory. Research funds were almost nonexistent, and even if we had had money, there were no sources of chemicals, experimental animals, or other needed commodities. The facilities were outdated, and the supply of electricity, gas, and even water was limited, so it looked almost impossible to start any experimental research. One day, I was visited quite unexpectedly by Dr. Yashiro Kotake, Professor Emeritus of Osaka University and a world-renowned biochemist in prewar Japan. He gave me several grams of tryptophan as a gift and encouraged me to use this amino acid for my research. I was very grateful to him but, frankly, did not know what to do with this precious material. At that time, tryptophan metabolism had already been investigated by numerous biochemists, including Kotake and co-workers. Tryptophan had been shown to be metabolized to kynurenic acid, anthranilic acid, and xanthurenic acid and secreted into the urine of rats and other mammals, including humans. Kotake was famous for this discovery and for the identification of kynurenine, the key intermediate of tryptophan metabolism. In 1934–1935, he wrote two solicited review articles on this subject in the Annual Review of Biochemistry (Volumes 3 and 4). Because Kotake's lifework had been carried out with the animal systems and because of the poor existing conditions of our laboratory, I decided to try microorganisms. I went out into the back yard of the University building, took a spoonful of scorched soil, mixed it with a trace amount of Kotake's tryptophan and water in a test tube, and simply waited. Several days passed, and then a faint white cloudiness appeared in the supernatant in the test tube containing tryptophan, whereas nothing happened in the test tube without the amino acid. This cloudiness slowly increased after several days, and by transferring the contents to another test tube containing tryptophan, I was eventually able to isolate a strain of soil bacteria that could grow with tryptophan as its sole source of carbon and nitrogen. This microorganism was later identified as a member of the genus Pseudomonas. I then found rather serendipitously that in this microorganism, unlike in mammals, anthranilate was further converted to catechol and then to muconic acid, which eventually decomposed completely to CO2, NH3, and H2O. Encouraged and excited by this somewhat unexpected discovery, I then tried to extract enzymes from this microorganism to study the details of this new metabolic pathway of tryptophan. The only enzyme that I was able to solubilize from the dried cells of Pseudomonas was the one that catalyzed the oxidative cleavage of the benzene ring of catechol, yielding cis,cis-muconic acid as the reaction product (Fig. 1) (1Hayaishi O. Hashimoto Z. J. Biochem. (Tokyo). 1950; 37: 371-374Crossref Scopus (65) Google Scholar). This enzyme had two unique and important features that had not been described before. 1) Unlike many other enzymes found in the past that metabolize phenolic compounds, it cleaved and opened the aromatic structure and produced an aliphatic compound. 2) Molecular oxygen could not be replaced by any other oxidant, electron, or hydrogen acceptors such as dyes, including methylene blue, dichloroindophenol, and so forth, or by coenzymes such as heme, FAD, NAD, etc. Various other properties of this enzyme appeared to be different from those of classical dehydrogenases and oxidases. A stoichiometric utilization of 1 mol of oxygen was demonstrated by the use of the Warburg respirometer, and so I naively assumed that molecular oxygen had been incorporated into the substrate, catechol. However, I was startled to learn later that the direct addition of oxygen had been completely ruled out in biological oxidation processes according to Professor H. Wieland, a Nobel Laureate in chemistry and the author of the book On the Mechanism of Oxidation, published in 1932 (2Wieland H. On the Mechanism of Oxidation. Yale University Press, New Haven, CT1932Google Scholar). According to his "dehydrogenation theory," the principle of biological oxidation is the activation and transfer of hydrogen atoms or their equivalents, in which case molecular oxygen may serve only as the hydrogen acceptor and be reduced to H2O or H2O2, but would never be incorporated directly into the substrate. Nevertheless, all available evidence in my hands was consistent with the conclusion that this enzyme was an oxygen transferase rather than a dehydrogenase or an oxidase. Therefore, I decided to propose to name this novel enzyme "pyrocatechase" rather than catechol oxidase (1Hayaishi O. Hashimoto Z. J. Biochem. (Tokyo). 1950; 37: 371-374Crossref Scopus (65) Google Scholar). However, when I spoke to my mentors and colleagues at Osaka University, most of them were skeptical of my interpretation because, at that time, the dehydrogenation theory of Wieland was an indisputable central dogma in the textbooks of biochemistry and enzymology. If I had proposed the direct addition of molecular oxygen to a substrate in my paper, it would not have been accepted by any reputable journal. In fact, in many textbooks published later, pyrocatechase long remained an orphan enzyme and was classified as miscellaneous or unclassified in a corner of the category of oxidoreductases (3Hoffman-Ostenhof O. Enzymologie. Springer-Verlag, Vienna1954Crossref Google Scholar). Thus, I did not mention clearly the additive oxygenation mechanism but simply stated the above-mentioned experimental observations and sent the manuscript to the Japanese Journal of Biochemistry, the only journal of this kind published in English in Japan at that time. The paper was accepted immediately without any problem. It was the most inexpensive piece of work in my life, perhaps almost as inexpensive as a seed of persimmon! While I was preparing the manuscript for publication, I sent a copy to Professor D. E. Green and asked him for suggestions and comments on my "enzymic oxygenation" hypothesis. I had never met him before, but I was aware that he had spent many years at Cambridge University, England, where Sir Frederick Hopkins had discovered tryptophan and investigated its metabolism and function, and that Green had recently returned to the United States to become the director of the newly built Enzyme Institute at the University of Wisconsin in Madison. I did not hear from him for several weeks and almost forgot about it. One day in the early spring of 1949, again fate directed a turn in my path. I received a letter from Green; he did not say much about my manuscript but instead politely invited me to come to the Enzyme Institute and offered me a William Waterman Fellowship in Enzyme Chemistry. I was pleasantly surprised. However, Japan was still an occupied country, and I was not sure if it would be safe for me in the United States and if I could possibly live comfortably in a foreign country that had been an enemy until a few years before. Indeed, most of my friends and colleagues cautioned me to wait several years until the situation had improved. After pondering over Green's letter for several days and again discussing the situation with my father, I finally decided to take a chance. I accepted his invitation. In November 1949, Green sent me an airplane ticket, and I departed from Haneda Airport in Tokyo on a double-decker four-engine Boeing B-377, which was a luxury in those days. After changing flights several times, I finally landed at the Madison airport in the middle of a snow flurry and was greeted by two postdoctoral fellows from Green's lab, Bernard Katchman and Ephraim Kaplan, both of whom later became my lifelong and best friends. In fact, they were the kindest and most considerate human beings I had ever met and treated me like a real brother, not only in the lab, acquainting me with various new methods and materials and jargon, but also in daily life, teaching me English, sometimes even Yiddish jokes, and introducing me to a Jewish delicatessen, which I liked very much. Thanks to their warm and generous hospitality and kindness, I was quickly able to adjust to the new environment despite a language barrier and the ethnic and religious differences and to enjoy the American way of life in this beautiful and peaceful university town. Soon I made many friends such as Philip Cohen, Henry Lardy, Van Potter, Takeru Higuchi, and others. Henry Lardy was especially encouraging and helpful to me and was interested in my "oxygenation hypothesis." He suggested that I should read several books and reviews that were pertinent and useful when I took up this problem several years later (4A Symposium on Respiratory Enzymes. University of Wisconsin Press, Madison, WI1942Google Scholar, 5Lardy H.A. Respiratory Enzymes. Burgess Publishing Co., Minneapolis, MN1949Google Scholar). In the meantime, I started to work on Green's cyclophorase hypothesis under his guidance and soon became aware that many of my own and other collaborators' experiments did not support his thesis. In fact, his cyclophorase hypothesis became controversial in the biochemical society at large. In April 1950, I attended the Federation meetings at Atlantic City and by chance listened to the paper given by Arthur Kornberg. It was a short report in the General Session lasting perhaps only 10 or 12 min, but I was so impressed and inspired by his presentation that I asked Bernard Katchman, who was sitting next to me, about Arthur. He told me that Arthur was a rising young star in biochemistry in the United States and advised me that if I wanted to remain in America another year or so, I should apply to work with a young mentor like Arthur. A few weeks later, Arthur was invited to Madison for a lecture, and I was able to meet him in person. Boldly, I asked him if he could take me into his lab as a postdoctoral fellow. He was very receptive to this idea and suggested that I should apply for a National Institutes of Health (NIH) fellowship, which I immediately did according to his instructions. While I was waiting to hear from NIH about my application for a fellowship, I received a telephone call from Professor Roger Stanier of the University of California; he invited me, with some urgency in his voice, to come to his laboratory for a collaborative project on tryptophan metabolism. He had previously and independently used the enrichment culture technique and isolated a pseudomonad from soil, but his strain degraded tryptophan via kynurenic acid instead of anthranilic acid. He proposed to isolate and purify all the enzymes involved in these two different pathways, which he named the "quinolinic" and "aromatic" pathways, respectively. Because his proposal was a very interesting and challenging one and was closely related to my previous experience in Osaka, I accepted his offer; so with Dr. Green's consent, I resigned my post at the Enzyme Institute, bid farewell to Bernie and Eph, and moved to Roger's lab in the Life Science Building at Berkeley at the end of August 1950. There again, I was fortunate to become acquainted with many outstanding scientists such as C. B. van Niel, H. A. Barker, Michael Doudoroff, William Hassid, and others, all of whom were kind, helpful, and welcoming. In Roger's lab, we both worked very hard day by day, exhausting almost all available methods for extracting enzymes from bacterial cells described in the textbooks. In fact, Roger stated, "I have never worked so hard before and after" (6Stanier R.Y. Annu. Rev. Microbiol. 1980; 34: 1-48Crossref PubMed Scopus (23) Google Scholar). Almost 3 months had passed without any sign of progress. Then one day I met H. A. Barker in the corridor and started casually chatting with him about my problem. He suggested that I try an old method described by Mirick some years ago in which alumina powder and bacterial cells are mixed and ground in a mortar with a pestle (7Mirick G.S. J. Exp. Med. 1943; 78: 255-272Crossref PubMed Scopus (5) Google Scholar). We were at first a bit skeptical because it sounded too simple and primitive; after all, the more sophisticated methods we had tried thus far had been uniformly unsuccessful. As a last resort, we tried it anyway. Lo and behold, it worked! We were able to extract almost all enzymes from the cell body and to partially purify them for further characterization. During the next month or so, Roger and I worked day and night and were able to publish a series of six papers over a span of about a month, which appeared in reputable journals such as the Journal of Bacteriology, Journal of Biological Chemistry, and Science (8Stanier R.Y. Hayaishi O. Science. 1951; 114: 326-330Crossref PubMed Scopus (9) Google Scholar, 9Stanier R.Y. Hayaishi O. Tsuchida M. J. Bacteriol. 1951; 62: 355-366Crossref PubMed Google Scholar, 10Stanier R.Y. Hayaishi O. J. Bacteriol. 1951; 62: 367-375Crossref PubMed Google Scholar, 11Hayaishi O. Stanier R.Y. J. Bacteriol. 1951; 62: 691-701Crossref PubMed Google Scholar, 12Tsuchida M. Hayaishi O. Stanier R.Y. J. Bacteriol. 1952; 64: 49-54Crossref PubMed Google Scholar, 13Hayaishi O. Stanier R.Y. J. Biol. Chem. 1952; 195: 735-740Abstract Full Text PDF PubMed Google Scholar). It was indeed the most productive period in my career, at least quantitatively speaking (6Stanier R.Y. Annu. Rev. Microbiol. 1980; 34: 1-48Crossref PubMed Scopus (23) Google Scholar). Time had passed so quickly, and in December 1950, Arthur telephoned me and told me that my application had been approved. Takiko, my wife, and Mariko, my daughter, who was about 3 years old at the time, both of whom were living in Japan, joined me in Berkeley, and we then moved to Bethesda, MD. Bethesda was a very nice attractive residential area in the suburb of Washington, D. C., and we were all very happy there. Arthur was the Chief of the Enzyme Section, NIAMD, but unlike Green's lab, his was a small one, and Arthur was doing his own experiments with Bill Pricer, a senior technician. I was the only postdoctoral fellow working in his group. Bernard Horecker and Leon Heppel were the other senior members in the section, and they worked independently of Arthur. Arthur introduced me to nucleic acid and phospholipid chemistry and metabolism, and we worked together on the bacterial degradation of uracil using the enrichment culture technique. I also introduced the use of microbial enzymes to explore the metabolism of histidine, histamine, urocanic acid, etc., in collaboration with Herb Tabor and Alan Mehler. These studies were reviewed in "Special Techniques for Bacterial Enzymes: Enrichment Culture and Adaptive Enzymes" in Methods in Enzymology (14Hayaishi O. Methods Enzymol. 1955; 1: 126-137Crossref Scopus (8) Google Scholar). Every day from noon to 1:00 p.m., we attended the so-called luncheon seminar. Every one in his lab took turns in alphabetical order and gave a seminar on a selected paper of his choice. This lunch seminar, which was also attended by people from other sections and departments, including Herb and Celia Tabor, Terry and Earl Stadtman, Alan Mehler, Bruce Ames, Jesse Rabinowitz, Hans Klenow, and others, had one important characteristic. In many seminars and journal clubs that I had attended previously, the speakers normally reported facts and described the results and conclusions, almost like a copying machine. In contrast, this luncheon seminar in Arthur's lab was devoted to the discussions and criticisms of every detail of the strategy of the paper; it was a sort of exercise in maneuvers instead of just one in learning new results and facts. This unique training course became legendary and a tradition in many other laboratories, especially in those of the Kornberg school. For example, when I moved back to Japan in 1958, this type of luncheon seminar became a famous event and was nicknamed the "Hayaishi gymnasium or training school" at Kyoto, Osaka, and Tokyo Universities, where I served concurrently as the Chairman of their Departments of Biochemistry. Two years passed quickly, and one day Arthur told me that he had decided to move to St. Louis as Chairman and Professor of the Department of Microbiology at Washington University School of Medicine, one of the leading medical schools in the United States, and that he wanted me to come with him as an assistant professor. I felt very flattered and honored but was not able to accept his offer immediately because I had been in the United States only 3 years as a postdoctoral fellow and had no confidence in myself regarding teaching students and handling administrative duties and other chores in English. However, Arthur was enthusiastic and persuasive, and after talking things over with friends and my wife, Takiko, we finally decided to accept Arthur's offer and moved to St. Louis in late 1952. Life in St. Louis was also quite pleasant. I met many outstanding scientists there, including Carl and Gert Cori, Martin Kamen, Stanley Cohen, Oliver Lowry, and others, all of whom were kind and helpful. As expected, however, I had to spend much of my time teaching and working on grant applications and other chores, all in English. Thus, my productivity in the lab slowed down to some extent. Summer in St. Louis was notoriously hot and humid, so we took a short vacation in the Rockies in the summer of 1954. When we came back to St. Louis, I unexpectedly received a phone call from Dr. Sanford Rosenthal of NIH, who asked me if I would be interested in assuming the position of Chief of the Toxicology Section at NIAMD, NIH, in Bethesda. I felt very flattered and honored. After all, it was only my 5th year in the United States, I was only 34 years old, and this was an offer of a position equivalent to one that Arthur had occupied only 2 years ago. I thanked Dr. Rosenthal for thinking of me but told him that I was not a toxicologist by training and that I did not think I was the best qualified candidate for this position. Dr. Rosenthal laughed and told me that he was well aware that I had started as a microbiologist but that I was now a full-fledged biochemist-molecular biologist. He said that NIH wanted someone who could change the old-fashioned toxicology laboratory into a modern biochemical and molecular biological toxicology section. Furthermore, he assured me complete freedom in choosing the subject and approach of my research program if I accepted this position. Arthur congratulated me and encouraged me to accept this offer, so once again, we were on the move. In December 1954, my family and I returned to Bethesda, which meant my fourth move during my 5-year stay in the United States! As I began to reorganize the research program of the Toxicology Section, I decided to investigate the mechanism of the pyrocatechase reaction because such a reaction might be involved in the detoxification of various drugs and poisonous compounds. I remembered that when I presented my previous work on pyrocatechase at the First Annual Meeting of the Japanese Biochemical Society after World War II in April 1949 at Kyoto University, Professor Yashiro Kotake, who had given me tryptophan as a gift several years before, mentioned that when he was a postdoctoral fellow at the University of Königsburg, Professor Max Yaffe, his mentor, had fed benzene to a dog and isolated muconic acid from the urine. Kotake suggested that such a pyrocatechase-like enzyme might also be present in mammals. Because 18O, a heavy isotope of oxygen, was not commercially available at that time, I wrote to Dr. David Samuel, head of the Isotope Department at the Weizmann Institute of Science in Israel, who kindly provided me with concentrated H 182O. We generated 18O2 by electrolysis and carried out the crucial but simple experiments. The results were clear-cut and demonstrated unequivocally that the oxygen atoms incorporated into the product of the reaction, i.e. cis,cis-muconic acid, had been derived almost exclusively from molecular oxygen, not from oxygen in water molecules (15Hayaishi O. Katagiri M. Rothberg S. J. Am. Chem. Soc. 1955; 77: 5450-5451Crossref Scopus (229) Google Scholar). A full account of these results, together with those of another set of experiments with 18O and H 182O conducted using tryptophan and tryptophan pyrrolase, was then published subsequently (16Hayaishi O. Rothberg S. Mehler A.H. Saito Y. J. Biol. Chem. 1957; 229: 889-896Abstract Full Text PDF PubMed Google Scholar, 17Hayaishi O. Katagiri M. Rothberg S. J. Biol. Chem. 1957; 229: 905-920Abstract Full Text PDF PubMed Google Scholar) and reviewed recently in Classics of the Journal of Biological Chemistry by Kresge, Simoni, and Hill (18Kresge N. Simoni R.D. Hill R.L. J. Biol. Chem. 2005; 280: e44Abstract Full Text Full Text PDF Google Scholar). These results therefore clearly established that, contrary to the central dogma proposed by H. Wieland, oxygen fixation reactions do indeed occur in biological systems. Concurrently and independently, H. S. Mason and co-workers reported that mushroom phenolase incorporated one atom of molecular oxygen into the substrate, whereas the other atom was reduced to H2O (19Mason H.S. Fowlks W.L. Pterson E. J. Am. Chem. Soc. 1955; 77: 2914-2915Crossref Scopus (170) Google Scholar). In 1956, I organized the first symposium on oxygenases at the American Chemical Society meeting in Atlantic City and proposed to name this novel group of enzymes that catalyze the incorporation of molecular oxygen into their substrate, viz. oxygen fixative reactions, as "oxygenases." In 1957, Professor Otto Hoffmann-Ostenhof of the University of Vienna came to visit me at my NIH office and invited me to organize and chair the first international colloquium on oxygenases at the 4th Congress of the International Union of Biochemistry (IUB), which was to be held in Vienna in 1958. It was my first association with the IUB, and I gladly accepted his invitation. In late 1957, Kyoto University School of Medicine decided to appoint me as Chairman and Professor of the Department of Medical Chemistry, one of the oldest and most prestigious centers of biochemistry in major Japanese universities at that time. In the same year, I was invited to attend the First International Meeting on Enzyme Chemistry held in Tokyo and Osaka as a delegate of the American Biochemical Society. After the meeting, I was able to spend several more weeks in Japan and had a chance to talk to a number of leading biochemists, my old colleagues, and friends and also met numerous young investigators, all of whom enthusiastically asked me to come back to Japan. After having returned to Bethesda and talking it over and over again with Takiko and my now 11-year-old daughter, Mariko, I finally made up my mind to accept the offer from Kyoto, and so we went back to Japan in February 1958. In the summer of that year, I went to Vienna to attend the 4th IUB Congress and chaired and presented a lecture in the Colloquium on Oxygenases, the first international meeting for this novel respiratory enzyme. After the Congress, I was invited to a number of universities and institutes in Europe and spent almost a month touring, lecturing, and meeting famous biochemists whose names had been known to me only from their publications and correspondence. I also learned a lot by making many new friends such as Sune Bergström, Bengt Samuelsson, and their associates, who told me about prostaglandins, and we discussed the oxygenase nature of prostaglandin synthase. In 1962, Oxygenases, the first comprehensive treatise on this subject, was published by Academic Press (20Hayaishi O. Oxygenases. Academic Press, New York1962Google Scholar). In 1964, I presented a plenary lecture entitled "Oxygenases" at the 4th IUB Congress in New York, which was chaired by Professor John T. Edsall, then Chief Editor of the Journal of Biological Chemistry. I still remember his kind compliments and warm words of encouragement after my talk. Needless to say, I was deeply touched. When I started to work in Kyoto in February 1958, Yasutomi Nishizuka applied to join me as my first graduate student. He later became one of the most famous biochemists in Japan after his discovery of protein kinase C. Soon the department was filled with young and ambitious postdoctoral fellows, graduate students, and visiting scientists from all over Japan as well as from abroad. All these people were bright, highly motivated, and hard working, and all were eager to learn dynamic biochemistry and enzymology, which I had learned from my experience during the past 10 years in the United States, especially from Arthur Kornberg. The time spent with Arthur, the first 2 years as a postdoctoral fellow in Bethesda and the subsequent 2 years as an assistant professor in St. Louis, provided valuable and unforgettable experiences for me, and our friendship lasted the rest of his life. Arthur Kornberg passed away in October 2007. However, the economical conditions in Japan were still lagging far behind. The exchange rate was ∼400 yen/dollar compared with the present rate of 105 yen/dollar. My salary as the youngest professor of Kyoto University was less than 8% of my salary at NIH. Fortunately, the Japanese Government had made a special effort to provide me with a large number of grants, and many foundations and pharmaceutical companies offered their generous support in the form of startup grants. In addition, NIH provided a substantial amount of money in the form of a research grant, and the Jane Coffin Memorial Fund, Rockefeller Foundation, China Medical Board, and several pharmaceutical companies in the United States contributed significant amounts of money as well, not only for research but also for rebuilding and remodeling the old buildings and even some for constructing a new building for radioactive experiments and a library. At first, oxygenase-catalyzed reactions were generally thought to be rather unusual and suggested to be limited to primitive forms of life such as soil bacteria and mushrooms. However, subsequent work in my Kyoto laboratory and experiments by others all over the world revealed that oxygenases are found ubiquitously in animals, plants, and microorganisms and play important roles not only in the biosynthesis and degradation of natural compounds but also in the degradation of synthetic compounds such as drugs, insecticides, chemicals, toxins, and so forth, as exemplified by cytochrome P450, now often described as the most versatile biological catalyst known (21Coon M.J. J. Biol. Chem. 2002; 277: 28351-28363Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In contrast, oxidases and dehydrogenases are mainly, if not exclusively, involved in energy metabolism. Fig. 2 shows a metabolic map of tryptophan, in which red oxygen atoms indicate the molecular oxygen incorporated into substrate by various specific oxygenases (22Hayaishi O. Protein Sci. 1993; 2: 472-475Crossref PubMed Scopus (28) Google Scholar). It illustrates the ubiquitous presence of oxygenase-catalyzed reactions in this physiologically important metabolic pathway and also shows the presence of numerous novel enzymes and metabolic reactions initiated by various specific oxygenases. For example, we were able to demonstrate the presence in cell nuclei of a new macromolecule, poly(ADP-ribose), which is synthesized from NAD and is found covalently attached to histones and other nuclear proteins (23Nishizuka Y. Ueda K. Nakazawa K. Hayaishi O. J. Biol. Chem. 1967; 242: 3164-3171Abstract Full Text PDF PubMed Google Scholar). Poly(ADP-ribose) is now known to participate in DNA repair, apoptosis, and chromatin stabilization. These studies were extended to elucidate the mode of action of diphtheria toxin. We discovered that diphtheria toxin catalyzed the ADP-ribosylation of aminoacyltransferase II, and this finding provided the molecular basis for the understanding of the toxicity of this toxin (24Honjo T. Nishizuka Y. Hayaishi O. Kato I. J. Biol. Chem. 1968; 243: 3553-3555Abstract Full Text PDF PubMed Google Scholar). This was the fi
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