My Contributions to Science and Society
2005; Elsevier BV; Volume: 280; Issue: 17 Linguagem: Inglês
10.1074/jbc.x400013200
ISSN1083-351X
Autores Tópico(s)Evolution and Genetic Dynamics
ResumoNot too many of my scientific colleagues have lived, as I have, through the birth pangs of a new state or felt the need to throw themselves into a lifestyle that is critical for their own survival and their nation's future. If this sounds dramatic, it is no more and no less than what it was like to be a scientist in the emerging and newly established State of Israel for the larger part of the 20th century when the local Jewish population and many Zionists living abroad were devoting all their energies to achieving statehood and then building and protecting the new state after its creation in 1948. Perhaps I may therefore be forgiven if these reflections on my scientific activities are inextricably interwoven with recollections of my life outside science. I have participated in the most significant events in my country during the historic period of its emergence and development as a dynamic new state. At the same time, I have derived enormous pleasure and fulfillment from my chosen path of research and teaching in the life sciences. In 1922, when I was 6 years old and my brother Aharon was 9, my family emigrated to Palestine from Poland. Our first home was Tel Aviv, then a tiny city taking shape on the sand dunes adjacent to ancient Jaffa. After a year we moved to Jerusalem. My brother and I were especially drawn to the natural sciences, and after high school we both decided to continue our studies at the new Hebrew University of Jerusalem. I began in 1932, 2 years after Aharon had enrolled in the university's first group of biology students. The ascent to Mount Scopus each day on my motor bike, one of the few motorized vehicles in Jerusalem in those days, was always an exhilarating experience with the Old City in front of me flanked by the mighty Judean desert to the west and the stone-colored new city shining in the east. Already in high school it was clear to me that, like all those of our generation, we would have to play our part in activities that had nothing to do with learning but were bound up with the national renaissance. Growing up in Palestine under the British mandate, and especially on the university campus, I was caught up in the ideological and political ferment of that time. Jews were returning to their ancient homeland after 2000 years, filled with the desire to build a democratic state in which we could determine our own future, revive our original language, and revitalize our culture. We were ready to forge a new society, which would be based on the principles of social justice defined by our biblical prophets and would offer a high quality of life enriched by the highest moral and spiritual values. In this exhilarating atmosphere, we threw ourselves with great enthusiasm into activities aimed at fulfilling the Zionist dream. In the 1930s and 1940s the local Arab population, angered by the increasing Jewish presence, often attacked the Jews. We had to protect ourselves, and this we did by joining the illegal Jewish defense organization, the Haganah, which later became the Israel Defense Forces. Thus, while still a student, I had already formed quite a clear idea of my goals in life. I would do what I could to help establish the State of Israel and contribute to its security and its social and economic development. In addition, I would attempt to do some original research while at the same time playing my part in raising a new generation of Israeli scientists and helping to create the physical and intellectual conditions in which science and technology could flourish in this region. Like Chaim Weizmann, whose life and work served as an inspiration to many young scientists, I believed with all my heart "that science will bring peace to this country, renew its youthful vigor and create the sources for new life, both spiritually and materially." I have been lucky enough to spend my life in pursuit of my goals, with some success and considerable satisfaction. The international tone of Israeli scientific endeavor was set in the early years of the Hebrew University by the excellent teachers, some of them world famous scientists, who had made their way to Palestine from the great centers of learning in England, Europe, and the United States. After years of solitary research before regular teaching activities started at the university, they were delighted that at last they had someone to teach. They treated their small groups of students as their friends and future scientific heirs, doing their best to endow us with all their accumulated knowledge. Professors and students roamed the country together, exploring and recording its flora and fauna, geology, water, and mineral resources. Our mathematics teacher was Binyamin Amira, who participated in the setting up of the Institute of Mathematics at the university and grew roses in his spare time. Shimon Samburski taught us physics, and Moshe Weizmann, Chaim Weizmann's younger brother, taught us organic chemistry. Leo Picard taught us geology and paleontology. I remember him handing me his book in English, dealing with the formation of the Dead Sea, with instructions to be ready to discuss its contents after 2 weeks. With Tchorna Reiss, a Romanian botanist who asked me to translate her lectures into Hebrew, I spent many hours at the swampy Lake Huleh near the Sea of Galilee, looking for plankton species, which I then described in my first scientific publication. I was captivated by the variety of exquisite unicellular organisms in the lake, the ordered patterns of their lives, and the wonderfully delicate silicon structures that some of them built for themselves. Alexander Eig, who headed the Department of Botany, was a self-taught expert on botanical ecology with an astonishing knowledge of the plants of Israel. He would take us into the Judean Desert on lengthy field trips, pointing out plant societies and describing their struggles as they competed with one another for survival. I well remember how the desert came to colorful life after a brief rainstorm, with myriads of plant species springing into flower. My enchantment with those unforgettable vistas led me to study the desert flora, and my second publication, together with my friend Gideon Orshan, was on the plants that survive in this arid area. Together with Haim Shifroni, headmaster of the school in Kibbutz Ein Harod, I also published two volumes on organic chemistry, which were used as high school textbooks for many years. Michael Evenari introduced my brother and me to plant physiology, a new and fascinating field of study. Gladly putting aside classification and collection and memorization of details, we tried instead to fathom the secrets of biological processes and the physical and chemical mechanisms that cause them. We became close friends of the zoologist Shimon Bodenheimer, with whom Aharon wrote a small book in Hebrew about the butterflies of Israel, called Children of the Sun. I can still see my brother running after butterflies and hardly ever catching one. I soon found myself under the spell of the biological sciences, with botany, zoology, and bacteriology as my major subjects. In trying to learn something about the structure, function, and behavior of these living organisms, however, I realized that I would first have to study chemistry, physics, and mathematics, and so I spent some years in the exact sciences before returning to biology. Here my interest was attracted by the large molecules, the macromolecules of the cell, which play a critical role in determining life processes. I was fascinated by the lectures of our biochemistry professor, Andor Fodor, who introduced me to the world of biopolymers. Most intriguing was the revelation that proteins not only constitute the basic building blocks of elaborate cellular structures but also act as molecular machines that carry out a multitude of complex reactions within cells and tissues. The research for my M.Sc. and Ph.D. degrees was done in the Department of Theoretical and Macromolecular Chemistry, headed by the late Max Frankel. Aharon, Frankel's laboratory assistant, was using potentiometric techniques to investigate the interaction of amino acids and peptides with aldehydes and sugars. Understandably, he persuaded me that for my master's thesis I should prepare salt-free basic trifunctional amino acids and investigate their electrochemical properties. These amino acids were not available on the market, so I had to prepare them from red blood cells. For about a year I collected blood from the slaughterhouse, separated and hydrolyzed the red cells, and isolated the basic amino acids lysine, arginine, and histidine from the hydrolysate by means of an elaborate electrophoretic technique. I needed amino acids for my doctoral research as well and was greatly relieved to discover that it was now possible to buy them. We also kept a low profile regarding our activity with the Haganah. I became an officer in this underground organization and for a while commanded a field unit but was mainly involved, with Aharon and others, in the establishment of the scientific research team that later became the Israeli army's research and development unit. One of the most useful books I came across during my graduate studies was Proteins, Amino Acids and Peptides as Ions and Dipolar Ions (by Edwin J. Cohn and John T. Edsall, published in 1943), which made me realize that to know something about proteins, I would first need to understand the structure and properties, in the solid state and in solution, of various high molecular weight proteins and polypeptides. Aharon and I spent many pleasant hours together in a small grove of trees outside the laboratory poring over whatever articles on polymer chemistry we could lay our hands on, and soon we could practically recite by heart the pioneer works of Hermann Staudinger, Herman Mark, Kurt Meyer, and Paul Flory. Within a year we were the undisputed experts on macromolecules in Palestine and within another year or two found ourselves leading the field in the Middle East. However, we felt completely isolated from the mainstream of scientific activity in Europe and the United States. Naturally there was a certain satisfaction in having one's own ideas uncontaminated by those of others, but this feeling was rapidly superseded by the need to exchange ideas with colleagues working in related areas. Staudinger, Meyer, and others had suggested that synthetic high molecular weight compounds might serve as useful models in the study of biopolymers. This idea caught my attention. Israel's plastics industry did not yet exist, and so the only available polymers were the polyethylene, polystyrene, nylons, and bakelite that we had to purchase for our laboratory. Although it was fascinating to realize that the structurally complicated plastic materials appeared to have multiple potential uses, for example as fibers, fabrics, and kitchenware, it was disappointing to find that they were biologically inert and therefore of no interest to biologists. I therefore set out to transform inert synthetic polymers into biopolymers that would be of biological relevance. What interested me was the synthesis of the simplest polymer, composed of repeats of one amino acid only. I assumed that if I could synthesize this macromolecule it might be possible, by studying its properties, to learn something about structure-function relationships in proteins. Also, it seemed to me that by covalently attaching amino acids, peptides, and proteins to selected inert synthetic polymers, it should be possible to endow these polymers with specific biological characteristics. The project seemed to be worth a try. At the start, I believed it would be possible to prepare amino acid polymers by carrying out well chosen polycondensation reactions of the corresponding amino acid esters. My results with this approach were not particularly impressive, and so I looked around for other amino acid derivatives that might yield the desired polymers. To my great satisfaction, I found that α-N-carboxyl amino acid anhydride, which Leuchs had prepared in 1906 and which can by now be readily prepared by interacting amino acids with phosgene, had yielded a reactive labile monomer that readily yielded the desired polyamino acid in the solid state or in solution. As I was particularly interested in preparing a basic polyamino acid, I decided to start with the synthesis of poly-l-lysine. I believed that the synthesis of this basic polyamino acid would shed new light on the chemical, biophysical, and biological properties of basic proteins such as the protamines and histones, which are found in all cell chromosomes in combination with DNA and seem to protect and regulate the activity of genes during development of the cell. The preparation of poly-l-lysine was finally achieved by polymerization of ϵ-N-carbobenzyloxy-α-N-carboxy-l-lysine anhydride to yield poly-ϵ-N-carbobenzyloxy-l-lysine and the removal of the protecting carbobenzyloxy group with phosphonium iodide, work carried out with my student Izhak Grossfeld. At first we assumed that the benzyl groups of the benzyloxycarbonyl residue are reduced by the liberated phosphine; however, as we found ourselves weeping copiously during synthesis, we realized that benzyl iodide was evolving as a result of the HI liberated. Many years later, these findings led Arieh Berger and Dov Ben Ishai in my laboratory at the Weizmann Institute to develop the classic technique for removal of the benzyloxycarbonyl-protecting groups with HBr in glacial acetic acid. When we sent in our first paper on the synthesis of poly-l-lysine to the Journal of the American Chemical Society, it was rejected by the editor, who was not convinced that a polymer had actually been produced. More hard work in the laboratory yielded evidence that persuaded even the most skeptical editor that what we had was indeed a high molecular weight, water-soluble polymer of l-lysine. I was delighted that we now had our synthetic macromolecule that showed all the characteristics of a high molecular weight polymer, and in the case of poly-l-lysine, of a polyelectrolyte as well. It was also gratifying to find that our poly-l-lysine was readily attacked by trypsin and interacted with viruses, bacteria, cells, and tissues in an interesting biological manner. The technique we developed opened the way for the preparation of linear homopolymers of other bi- and trifunctional amino acids, in which the steric configuration of the amino acid monomer was always retained during polymerization. In the meantime, our work at the Hebrew University was coming to an end as our proposed research budgets, each amounting to about $30 a year, were beyond the means of the university's treasury. Both Aharon and I were therefore in a receptive mood when Chaim Weizmann, the distinguished organic chemist who in 1948 became the first President of the State of Israel, invited us in 1946 to join the academic staff of the new scientific center to be named after him. The planning committee of the new Weizmann Institute of Science was headed by Ernst David Bergmann, Weizmann's distinguished assistant, and Herman Mark, Head of the Polymer Institute at the Polytechnic Institute of Brooklyn, who in 1947 invited me to spend some time in his world famous Polymer Center. On the way there I spent a few weeks as a Research Fellow at Columbia University with David Rittenberg, learning the new isotopic labeling techniques for identifying and characterizing intermediate metabolites. Rittenberg was aware of my work on poly-l-lysine and drew my attention to a recently published short note by Robert Woodward and C. M. Schramm in the Journal of the American Chemical Society (1Woodward R.B. Schramm C.H. Synthesis of protein analogs..J. Am. Chem. Soc. 1947; 69: 551-552Crossref Scopus (65) Google Scholar). The title of their work, "Synthesis of protein analogs," gave me considerable satisfaction, as it clearly showed that Woodward and Schramm thought, just as I did, that poly-α-amino acids would be useful as simple high molecular weight models for proteins. Herman Mark organized the purchase of the first sophisticated scientific equipment for the Weizmann Institute—an ultracentrifuge, an electron microscope, an electrophoretic apparatus, and an x-ray diffractometer. Palestine at that time (1947) was in turmoil, with the British preparing to leave and our leaders girding themselves for the declaration of the State of Israel. Rather than risk shipping our precious hardware to Rehovot, Mark had it temporarily installed in the laboratories at Brooklyn. He even suggested running the Weizmann Institute as part of the Brooklyn Polytechnic until things settled down, an offer I naturally declined, and within a short time the equipment and I were home in Rehovot. This was at the beginning of May 1948. Most of my colleagues were by now involved in intensive on-campus research and development activities for the Haganah. Other types of research were virtually at a standstill. Aharon and I threw ourselves into whatever had to be done, drawing on all our professional expertise to assist in the defense of the new state. It was painfully clear to all of us that, much as we might aspire to careers in basic research, survival was the first necessity. The State of Israel was established on May 14, 1948. On the same day, the new State was invaded by five Arab armies and found itself fighting for its existence. I was temporarily placed in charge of the Israeli army's science corps, and until the end of the War of Independence we carried out military research, laying the foundations for the army's scientific defense unit, Hemed (an acronym for Cheyl Mada or Science Unit), which was established by Aharon, Yochanan Ratner (from the Technion in Haifa), Ernst David Bergmann (by then head of the Weizmann Institute), and myself. Most of the scientists at the Institute were in uniform, laboratories were in use day and night, and the formerly tranquil campus resounded with the test explosions of new weapons. What we lacked in arms experience we made up for in motivation and a talent for innovation, and this work prepared the way for Israel's future defense industry. We designed and produced various items of defensive equipment. Just prior to the establishment of the State, Ben-Gurion had taken upon himself the position of unofficial defense minister. I remember that while still in Brooklyn I received a letter from my brother describing his meeting with Ben-Gurion, who had summoned him to hear about Hemed's activities and to offer his help. Aharon told him that the unit needed money, whereupon Ben-Gurion reached in his pocket and handed him fifteen English pounds. Aharon was delighted, and wrote that they had hardly known what to do with the unit's new-found wealth! Nearly 60 years later, Rafael, which grew out of Hemed, is a billion dollar company producing highly sophisticated military equipment in cooperation with the Israel Defense Forces. During our War of Independence, some of the American scientists who were supposed to take charge of departments at the Institute became jittery about coming to Israel. Consequently, Aharon was asked to be temporary head of the Department of Polymers, and I was made acting head of the Department of Biophysics. These two appointments soon became permanent. Shortly afterward, in 1951, at the invitation of John Edsall, I first came to Harvard University and its medical school as a Visiting Scientist and have maintained close contacts with my colleagues there ever since. The department at that time was headed by Edwin Cohn. It took a while, I remember, to become familiar with the Harvard scene and style. After some months, having garnered the courage to come up with my own proposals for research, I would talk them over with John Edsall, who unfailingly encouraged my efforts. Next I would call on Larry Oncley, who unfailingly discouraged them; he would assure me that my ideas could not work or had already been tried without success. My next sounding board was Edwin Cohn, who would enthusiastically collar me and deliver lengthy monologues on his own projects. At Harvard I established lasting friendships with Elkan Blout, Paul Doty, Bob Woodward, and Konrad Bloch, all of whom encouraged me to continue with my original research and offered useful critical comments. Poly-α-amino Acids as the Simplest of Protein Models—After moving to the Weizmann Institute, I continued to extend my work on polyamino acids as protein models. With my colleagues and students I synthesized several other polyamino acids, as well as amino acid copolymers and multichain polyamino acids, including branched macromolecules. By this time, other groups were also preparing polyamino acids and studying their properties: Mark Stahmann in Wisconsin (2Stahmann M.A. Polyamino Acids, Polypeptides and Proteins. University of Wisconsin Press, Madison, WI1962: 347Google Scholar), Clement Bamford in England, and Elkan Blout and Paul Doty at Harvard. Some of these synthetic polymers could be drawn into fibers whose conformation, as determined by x-ray analysis, resembles that of silk and wool keratin. The information gathered by Bamford's group at the research laboratory of Cortaulds in Maidenhead, Berkshire on the conformation and conformational transitions occurring in polyamino acids prompted the company to build a pilot plant for the production of poly-γ-methyl-l-glutamate fibers and cloth. I still have a piece of cloth made of these fibers, given to me on one of my visits to the British group. Because of the high costs of raw materials and production the project was dropped but not before a film studio had produced a movie starring Alec Guinness in an indestructible white flannel suit made of poly-γ-methyl-l-glutamate. The availability of high molecular weight polyamino acids opened the way to the x-ray analyses of poly-γ-benzyl-l-glutamate fibers done by Max Perutz in 1951, which confirmed the predictions of Pauling and Corey in connection with the α-helical polypeptide backbone. These data as well as the findings of Elliott and Malcolm in 1959 helped John Kendrew and Max Perutz decipher the x-ray patterns of myoglobin and hemoglobin. In addition, polyamino acids synthesized and studied in my laboratory by Arieh Berger, Joseph Kurtz, and Jurgen Engel served as useful models for elucidating the structure of collagen. With the availability of poly-α-amino acid models it was possible to clarify, during the 1950s and 1960s, the mechanism and kinetics of polymerization of N-carboxyl amino acid anhydrides, determine the α-helical conformation of some of the polyamino acids in the solid state and in solution, detect β-parallel and anti-parallel pleated sheets of polyamino acids, and induce helix-coil transitions in the solid state and in solution under appropriate conditions. Fruitful collaboration between experimentalists and theoreticians like H. Scheraga, J. Schellman, J. R. Tinoko, M. Levitt, and S. Lifson facilitated the successful correlation of the macromolecular conformations of polyamino acids in solution with their hydrodynamic properties, optical properties, dipole moments, and nuclear magnetic properties (3Katchalski E. Sela M. Silman H.I. Berger A. Polyamino acids as protein models.in: Neurath H. The Proteins. 2. Academic Press, New York1964: 405-581Google Scholar). Biological Properties of Polyamino Acids—Meanwhile, in Rehovot during the late 1940s, I concentrated on the study of the biological properties of polyamino acids. To my delight, poly-l-lysine and other homopolyamino acids and amino acid copolymers turned out to be excellent models for investigation of the mechanism of enzymatic protein hydrolysis and transpeptidation. I still remember the excitement with which I followed the rapid hydrolysis of poly-l-lysine by trypsin, using the cumbersome old Van Slyke apparatus. We showed that the specificity of an enzyme acting on a high molecular weight polypeptide is often strikingly different from that observed with low molecular weight peptides. Partial hydrolysis of poly-l-lysine yielded, as expected, a mixture of lysine oligomers. These were separated chromatographically and investigated immunologically by my former student Arieh Yaron, in Herb Sober's laboratory at the National Institutes of Health in the United States. Uptake of these oligomers by Escherichia coli was studied by Charles Gilvarg of Princeton University, then a visiting scientist at the Weizmann Institute. By using a lysineless mutant of E. coli, Gilvarg showed that E. coli readily takes up all oligomers up to tetralysine, but not larger polypeptides, and that these oligomers permit growth of the lysine auxotroph. In our experiments with a prolineless mutant of E. coli, my co-worker Sara Sarid observed that the organism can grow on a synthetic medium in which poly-l-proline is substituted for l-proline. Clearly, the polymer was being hydrolyzed by an unknown enzyme. Further investigations by Arieh Yaron of the cleavage of various synthetic proline-containing oligo- and polypeptides led to the identification and characterization of a novel enzyme, aminopeptidase P, in prokaryotes and eukaryotes. Antigenicity of Poly-α-amino Acids—An important outgrowth of the studies on synthetic polyamino acids was the development in my laboratory of techniques for the preparation of polypeptidyl proteins (proteins to which polypeptide chains are covalently attached via amide bonds to the free amino groups of the protein). The synthesis of polytyrosyl gelatin and the demonstration that it is antigenic, in contrast to the unmodified protein, led in 1960 to the preparation by Michael Sela and Ruth Arnon, then in my department, of the first fully synthetic antigen. In this compound, tyrosine and glutamic acid residues are attached to a multi-poly-dl-alanyl poly-l-lysine. I vividly remember our immunological experiments, in which guinea pigs injected two or three times with polytyrosyl gelatin went into anaphylactic shock. Besides being a nasty experience for the guinea pigs, this was a sobering demonstration to me of how careful one should be in treating living beings with synthetic or even native polymers. Nevertheless, the way was now opened for the fundamental and extensive studies of Sela and his co-workers on the chemical and genetic basis of antigenicity. Some of the polypeptidyl enzymes we prepared retained full enzymatic activity. This finding was the basis for our subsequent preparation of a great variety of immobilized enzymes (4Katchalski-Katzir E. Immobilized enzymes—learning from past successes and failures..Trends Biotechnol. 1993; 11: 471-478Abstract Full Text PDF PubMed Scopus (359) Google Scholar). Use of Poly-α-amino Acids in Deciphering of the Genetic Code—Knowledge of the properties of synthetic polypeptides played a decisive role in the work that led in 1961 to the cracking of the genetic code. In their first paper on the subject, Marshall Nirenberg and J. H. Matthei identified the poly-l-phenylalanine, produced enzymatically in a cell-free system in the presence of polyuridylate used as messenger, with the poly-l-phenylalanine we had synthesized in Rehovot. As it happens, Michael Sela was at NIH when Nirenberg was working on the code, and he had informed Nirenberg that the normally insoluble poly-l-phenylalanine could be dissolved in acetic acid saturated with HBr. Soon afterward, Nirenberg and Ochoa identified other homo- and heteropolyamino acids as part of the effort to decipher the genetic code: poly(A) was found to code for poly-l-lysine, poly-C for poly-l-proline, and poly-G for polyglycine. A Treatment for Multiple Sclerosis—Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system in which infiltrating lymphocytes, predominantly T cells and macrophages, cause irreversible damage to the myelin sheath. It is thought to be an autoimmune disease associated with an early viral infection. Based on previous clinical information, Michael Sela and Ruth Arnon examined the effect of a copolymer prepared in my laboratory, consisting of l-Ala:l-Glu:l-Lys:l-Tyr (6.0:1.9:4.6:1.0) on rats and mice suffering from experimental allergic encephalomyelitis, an animal model for MS. Their encouraging results led to development of the drug known as copolymer I (Cop-1), termed Copaxone and glatiramer acetate by the industry and widely used today as a therapeutic vaccine to reduce the rate of progress of MS in patients with the exacerbating-remitting form of this disease. Proteins with Glutamine Repeats and Reiteration of Other Amino Acids—Four neurodegenerative diseases are linked to excessive repeats of glutamine residues near the N terminus of affected proteins. They are Huntington's disease, spinobulbar muscular atrophy, spinocerebral ataxia type 1, and dentatorubral-pallidoluysian atrophy. The more numerous the glutamine repeats, the more severe the disease and the earlier its onset. The repeats tend to lengthen in successive generations of affected individuals, especially in male transmission. These findings prompted Max Perutz and his collaborators to construct molecular models of poly-l-glutamine and study their optical, electron, and x-ray diffraction properties. Their published data disclosed the presence of β-sheets strongly held together by hydrogen bonds, suggesting that glutamine repeats might function as polar zippers by joining specific transcription factors bound in separate DNA segments. In line with these findings an impressive set of data on codon reiteration, published by Green and Wang (5Green H. Wang N. Codon reiteration and the evolution of proteins..Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4298-4302Crossref PubMed Scopus (76) Google Scholar) showed that hydrophobic amino acids, and particularly glutamine, accoun
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