Irwin Rose was one of the first scientists working on UPS mechanisms. In particular, his early work was dedicated to the biochemistry and the role of nonlysosomal protein degradation. But how did it all begin? What triggered his scientific interest in this field from his previous work? Now a large family of related proteins exist, with interesting therapeutical potential. Here, Cell Death and Differentiation asks Irwin Rose about the early work on enzymology. This interview was obtained, thanks to the kind help of the Nobel Foundation in Stockholm, http://nobelprize.org (©The Nobel Foundation 2004).
We left my birthplace, Brooklyn, New York in 1939 when I was 13. I enjoyed the ethnic variety and the interesting students in my public school, P.S. 134. The kids in my neighborhood were only competitive in games although unfriendly gangs tended to define the limits of our neighborhood. The major extracurricular activities that I can remember were a Victory Garden on school grounds, our contribution to the war effort, and a favorite sport, handball, played between the walls of our apartment house. Mother, Ella Greenwald, was American born into a family that included one sister and four brothers, all born in Hungary. Father, Harry Royze, had two brothers and a sister from the Odessa region of Russia. The Greenwalds and the Roses were secular Jews and the children more so although my younger brother and I spent some time in Hebrew school to please Grandfather Rose. Due to my brother having rheumatic fever the family was advised to go to a high and dry climate, Spokane, Washington, where my mother's sister had a comfortable home that could accommodate us. My father left behind to tend to his flooring business, an arrangement that I never understood and felt conflicted about. Father's visits were few and far apart. The war was going on and mother did secretarial work in the Navy Supply Depot in Spokane while we kids were making our way through the Spokane school system. I worked during the summers at a local hospital, chiefly helping out in the psychiatric ward. In time I came to see myself following some career that involved solving medical problems. No one in my family had followed a career in research. Uncle Arthur G was an excellent violinist and taught cabinet making at a trade school in Brooklyn and uncle Dave R would have become a lawyer had the economic depression not led him into the US Internal Revenue Service. There was no one in my circle from whom I could expect to get advice. Initially, I thought problems on how the brain works to be the most interesting. But it was necessary to be practical, and concentrate on less obscure matters when I entered Washington State College. Besides, there were no courses given in neurobiology. I then went to the University of Chicago after a brief period in the Navy. My Ph.D. thesis problem was to determine if the DNA content of rat tissues increased if there was B12 in the diet. This problem was suggested by my adviser based on the observation that thymine could replace vitamin B12 in a lactobacillus. I analyzed the DNA of tissues of rats fed diets that varied in B12. This project was doomed to failure when the genetic nature of DNA was revealed, and I found that the DNA content per cell of liver was independent of diet.1 I had to think of a new thesis project. Anxious to make up for lost time I picked a problem out of my Freshman biochemistry lecture notes. The Putnam/Evans group was interested in determining the origin of the nucleic acid components of bacteriophage synthesized in E. coli and Frank Putnam's lectures described experiments of Hammarsten, Reichard, and Saluste2 as background information. N15-labeled cytosine, the free base, had been found not to be incorporated into DNA although N15 of cytosine riboside was reported to be in rat liver DNA. It was obvious for me to ask if there might be direct utilization of the whole of cytidine, ribose and all, in the biosynthesis of deoxycytidine. That would be a shock. I learned from Peter Reichard, during the 2004 reminiscence in Stockholm, that the export to Sweden of C14 was forbidden by the AEC at that time; otherwise they certainly would have done the obvious follow-up experiment using U-C14-labeled cytidine themselves. I made RNA from Euglena gracilis grown on C14 labeled CO2. I had to work out the determination of the independent specific activities of the sugars and bases, which I did by treating the nucleosides with nucleoside phosphorylase and hypoxanthine to exchange for the base to be analyzed. Then by paper chromatography, using a medium containing borate to retard the migration of ribosides, I could also isolate deoxyinosine and cytosine. Although U-C14 cytidine did not label the deoxyribose of E. coli DNA, I found the deoxycytidine of DNA of rat organs to be almost uniformly labeled. The C14 content was far in excess of the negligible radioactivity in the purine deoxynucleotides.3 Therefore, on both counts it appeared certain that the C14 reached the deoxyribose directly from the cytidine. Reichard repeated and extended this experiment with U-C14 uridine in 1957 with much the same result. It would have been natural for me to try to work out the enzymology of ribonucleotide reduction after graduating. Peter Reichard at Yale on a post-doc from Sweden asked me about my intentions. But I was not anxious to get embedded in such a difficult problem at this early point in my career. I do not regret this decision since it may have facilitated Reichard's beautiful work. I was interested in learning more about the principals of enzymology. Ogston's 1948 paper proposing, in effect, that the ability of an enzyme–substrate complex to distinguish between identical groups on a tetrahedral carbon was a consequence of the asymmetry of the complex,4 was a matter of hot debate in chemistry/biochemistry circles at Chicago in 1950 where the enzyme was still a black box and the emphasis was on the chemistry of changes in the substrate. In particular, the Ogston idea could justify the conclusion in the experiments of Myron Bender, done in Chemistry at the University of Chicago, that the absence of back labeling of an ester in C18 water during enzymatic hydrolysis could not rule out a tetrahedral intermediate as could be inferred from the back labeling that occurred during alkaline hydrolysis.5 In the case of the enzymatic reaction one would expect to lose all the C18 on the stereospecific return of the intermediate to the ester. Thus, I became challenged to establish the absolute stereochemistry of enzymatic reactions and to determine its mechanistic significance, if any. This didn't seem such a formidable task although it was not until 1963 that Kenneth Hanson and I solved the historically important problem of the prochirality of citric acid,6 which was necessary to gain a proper perspective on the aconitate hydratase reaction. In 1955, after post-doctorals at Western Reserve University with CE, Carter and at New York University with Severo Ochoa, I was fortunate to be invited by Joseph Fruton to become an Instructor in Biochemistry at Yale University Medical School. The first year at Yale was notable for two developments. Not being willing to spend the time it would take to get the Department's mass spectrometer working, I turned to the scintillation counter, which was available in the medical school lab of Seymour Lipsky, an MD with a passion for exploiting new methods. One of the pioneer instruments to become available came from a small start up company in New Haven that Lipsky had been encouraging, the Technical Instrument Company. Lipsky also had a sample of tritiated water, which together with his counter got me started on experiments I wanted to do. The second development of great note in my first year was to propose to and marry Zelda Budenstein, a graduate student in the Department. Fortunately, I caught up with her before she graduated. Her mother, widowed since Zelda was age five, came to live with us. She was a great help with the four children that were in our onrushing future. This enabled Zelda to have a research career, often paralleling mine, which she continued until 1987 when she retired to devote full time to her peace and social interests.
