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Reminiscences of Leon A. Heppel

2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês

10.1074/jbc.x400007200

1083-351X

Leon Heppel,

Microbial Metabolic Engineering and Bioproduction

My parents were converted Mormons who had emigrated from Germany to Utah planning to live on a farm. The oldest of five children, I was born in Granger, Utah, in 1912. Farm life proved difficult and after 10 years our family moved to San Francisco. There, the city encouraged interesting local activities particularly for poor people, and life was more pleasant than in Utah. In school, I became interested in chemistry. While a high school student, my mother, who was ambitious on my behalf, persuaded John Stauffer, president of Stauffer Chemical Company, to give me a job doing analytical work at the American Cream Tartar Company in San Francisco. This supported me through high school and afterward when I enrolled at the University of California, Berkeley to major in chemistry and chemical engineering. Unhappily, my job at the American Cream Tartar Company and the support it provided did not last. In 1931, the Stauffer Chemical Company merged with the Schilling Spice Corporation and the combined company owned American Cream Tartar. A vice president of Schilling Spice undertook to effect economies, but the only economy he could find was getting rid of me. Shocked and urged by my mother to plead my case, I told the vice president how much I depended on the job. His cold reply was, “You need Schilling Spice Company but does Schilling Spice need you?” I never forgot those cruel words. Because of them, I abandoned my plan to be a chemical engineer turning instead to physiological biochemistry, which I thought would be a gentler profession. Fortunately I received a fellowship that allowed me to complete a B.S. degree in 1933. That same year I entered Berkeley's graduate school as a biochemistry student. Living in midtown San Francisco and commuting each day to Berkeley was a tiring chore. The Bay Bridges had not been built. In early morning, I took a streetcar to the Ferry Building where I boarded a boat for Oakland; on good days this took half an hour, but if the fog was intense, it was a much longer trip. From Oakland an electric train went to Berkeley and the university. In midafternoon, I returned across the Bay and spent a few hours working in one of the several Stauffer Chemical factories. Aside from the commute, however, life and science in Berkeley were exciting. During this period, Ernest O. Lawrence and others were doing great work and were anxious to talk about it. I made good friends among the chemists, one of whom discovered 14C (Martin D. Kamen in 1940). Nutrition was a major subfield of biochemistry in the 1930s, and I decided to do my thesis in that subject under Professor C. L. A. Schmidt. Schmidt was harsh and domineering but helpful. In later years when he became dean of the College of Pharmacy at the University of California, San Francisco, he hired my mother to take charge of equipment and supplies. For my thesis research, I decided to work on potassium (K+) metabolism in white rats. The experiments showed that K+ was essential for the growth and survival of young rats, and there was some evidence that sodium (Na+) could partially replace K+. Rubidium (Rb+) supported good growth in K+-free diets for a month, but thereafter the rats developed sudden tremors and died. My Ph.D. degree in biochemistry was awarded in 1937, a year when there were no jobs available for a biochemist. Luckily, Schmidt came to my rescue. He remembered a promise that George Whipple had made when he left Berkeley to start a new medical school in Rochester, New York. Whipple had told Schmidt that if he ever had a Ph.D. student who decided to come to medical school in Rochester, the student would receive partial support from the school. Right after receiving the Ph.D., I boarded a train for Rochester. Good fortune in the shape of a mentor came my way in Rochester. My work at Berkeley had attracted the attention of W. O. Fenn, a brilliant young physiologist who was a very quiet person and unusually kind. Fenn spent much of the day doing experiments with the help of a cheerful but somewhat talkative young woman. He gave me a position and suggested that I continue to study K+ metabolism in young rats. My initial results replicated my earlier finding that the rats grew well for a while when Rb+ replaced dietary K+ but then quickly developed tremors and died. In the early phase, although the rats appeared to be healthy, 7.5% of their muscle K+ was replaced by rubidium. Other experiments demonstrated that Na+ could replace K+ to some extent, and studies with radioisotopes confirmed that K+ and Na+ were able to cross an animal cell membrane. This was an astonishing finding, as German physiologists believed that the lipid cell membrane prevented passage of hydrophilic metal ions. Thanks to the generous spirit of Fenn, I was the sole author on three papers describing this work (1Heppel L.A. The electrolytes of muscle and liver in potassium-depleted rats.Am. J. Physiol. 1939; 127: 385Crossref Google Scholar, 2Heppel L.A. The diffusion of radioactive sodium into the muscles of potassium-deprived rats.Am. J. Physiol. 1940; 128: 449Crossref Google Scholar, 3Heppel L.A. Effect of age and diet on electrolyte changes in rat muscle during stimulation.Am. J. Physiol. 1940; 128: 440Crossref Google Scholar). By 1942 when I completed the M.D. degree and internship at Rochester, my work there had drawn considerable attention, and I received three offers for assistant residency positions from schools where interest in electrolytes was great: Yale Medical School with John Peters, Columbia University with Robert Loeb, and San Francisco Medical School. However, the entry of the United States into World War II interrupted normal, peacetime activities. Arthur Kornberg, a close medical school friend, and I joined the United States Public Health Service. Kornberg received sea duty while I was assigned to the National Institutes of Health (NIH). At NIH under orders from the Navy, I carried out tedious studies on the toxicity of halogenated hydrocarbons. Most importantly, the future began to take shape when I made a new friend, the enzymologist Bernard Horecker. Also, I persuaded Rolla E. Dyer, Director of NIH, to bring Kornberg to Bethesda. Together with Kornberg and Herbert Tabor and with the help of Horecker, I began to learn enzymology. Kornberg then left to spend a year (1946) in the laboratory of Severo Ochoa in New York and another (1947) with Gerty and Carl Cori in St. Louis. When he returned to NIH, he started a new research section for the study of enzymes and invited Horecker and me to join. Leaning on my background in toxicology, I began to examine the behavior of enzymes in toxic situations. Also, I investigated the metabolic reactions that convert inorganic nitrite to nitrate and nitroglycerines. I also purified inorganic pyrophosphatase and crystallized it with the help of Moses Kunitz (of the Rockefeller Institute (now University)) and purified 5′-nucleotidase. Then, in about 1951, my attention turned more generally to the phosphorylation and hydrolysis of purine ribonucleosides. This led, quite naturally, to an interest in enzymes that might hydrolyze RNA. Accordingly, my technician, Russell Hilmoe, and I purified from spleen an enzyme that partially solubilized RNA. The next step was to determine which linkages in RNA were split and which were resistant to the enzyme action. Roy Markham and J. D. Smith in Cambridge, England had demonstrated that fragments produced by RNA hydrolysis could be separated using paper chromatography and paper electrophoresis. Fortunately, I succeeded in obtaining a year's leave of absence from NIH, one of the first sabbaticals to be offered there, and spent a profitable year abroad in the laboratory of Markham. My work in England included the demonstration that the natural configuration of purine nucleotides in RNA was 3′–5′ rather than the alternative 2′–5′ (4Heppel L.A. Markham R. Hilmoe R.J. Natural configuration of the purine nucleotides in ribonucleic acids.Nature. 1953; 171: 1151Crossref PubMed Scopus (2) Google Scholar). Further evidence for this linkage was obtained from a study of the action of nucleases on mononucleotide esters carried out with Daniel Brown and Lord Alexander Todd (5Brown D.M. Heppel L.A. Hilmoe R.D. The action of some nucleases on simple esters of monoribonucleotides.J. Chem. Soc. 1954; 4576: 40Crossref Google Scholar). Also, the early steps in the hydrolysis of RNA by pancreatic ribonuclease were worked out in a collaboration with Paul R. Whitfeld (6Heppel L.A. Whitfeld P.R. Nucleotide exchange reactions catalyzed by ribonuclease and spleen phosphodiesterase. I. Synthesis and interconversion of simple esters of monoribonucleotides.Biochem. J. 1955; 60: 1Crossref PubMed Scopus (7) Google Scholar). This work lead to the isolation, by paper chromatography and paper electrophoresis, of cyclic terminal oligonucleotides. Whitfield, an Australian graduate student in the laboratory, was an excellent colleague in research and deserving of the credit he received when his name appeared on five of our publications (for example, Refs. 6Heppel L.A. Whitfeld P.R. Nucleotide exchange reactions catalyzed by ribonuclease and spleen phosphodiesterase. I. Synthesis and interconversion of simple esters of monoribonucleotides.Biochem. J. 1955; 60: 1Crossref PubMed Scopus (7) Google Scholar, 7Heppel L.A. Whitfeld P.R. Markham R. Nucleotide exchange reactions catalyzed by ribonuclease and spleen phosphodiesterase. II. Synthesis of polynucleotides.Biochem. J. 1955; 60: 8-15Crossref PubMed Scopus (22) Google Scholar, 8Heppel L.A. Whitfeld P.R. Markham R. A note on the structure of triphosphopyridine.Biochem. J. 1955; 60: 19Crossref PubMed Scopus (1) Google Scholar). Later on, I had an interesting interaction with Markham and Sutherland. Dr. Markham found that heating ATP with dilute alkali caused the formation of substantial quantities of a new compound whose properties puzzled him, as he related in a letter to me. At a later date, Dr. Sutherland wrote about a compound isolated from liver in minute quantities. It was biologically active. The two letters ended up in different parts of a pile of mail. However, one day I chanced to re-read both letters and I figured that these compounds were the same. This turned out to be so, and thus cyclic adenylic acid became readily available. I returned to NIH in January of 1954. Interesting and stimulating visitors began to come to the laboratory to learn techniques and collaborate. Henry Kaplan, a very distinguished Professor of Radiology at Stanford spent a sabbatical in the laboratory. Three joint papers were published with Horecker and Jerard Hurwitz, then a beginning researcher and now a distinguished biochemist. Jack Strominger was also a welcome visitor; the two of us, together with Elizabeth Maxwell, studied the phosphorylation of nucleoside monophosphates by nucleoside triphosphates. At this time, there was considerable interest in the results and methods I had obtained during my stay in England. A good deal of attention was being paid in particular to the demonstration that “synthetic” oligonucleotides could be synthesized by enzyme-catalyzed nucleotide exchange reactions (7Heppel L.A. Whitfeld P.R. Markham R. Nucleotide exchange reactions catalyzed by ribonuclease and spleen phosphodiesterase. II. Synthesis of polynucleotides.Biochem. J. 1955; 60: 8-15Crossref PubMed Scopus (22) Google Scholar). Before long, I learned about the discovery of polynucleotide phosphorylase in Azotobacter vinelandii by Marianne Grunberg-Manago and Ochoa at New York University. The same enzyme was independently discovered in Escherichia coli by Uri Littauer and Kornberg. At the time, I was one of only a few individuals who had the knowledge and experience required to study this enzyme and its products. Ochoa proposed that we collaborate and I accepted. Early in the course of the collaboration, a very able and pleasant postdoctoral fellow, Maxine Singer, joined my laboratory. She contributed greatly to the studies and made the association enjoyable. We put to good use all that I had learned in England about polyribonucleotides. One of our important findings was that short oligonucleotides could serve as primers for polynucleotide phosphorylase (9Singer M.F. Heppel L.A. Hilmoe R.J. Oligonucleotides as primers for polynucleotide phosphorylase.Biochim. Biophys. Acta. 1957; 26: 447Crossref PubMed Scopus (5) Google Scholar). Some time later, Singer and I used polynucleotide phosphorylase to prepare polyribonucleotides and oligoribonucleotides that Nirenberg used in his work on the genetic code. Singer continued to work on polynucleotide phosphorylase when she became an independent investigator. The elegant organic synthesis of oligonucleotides by Khorana was not available until a later period. Therefore, when working on the genetic code, it was an advantage to be able to use enzymatic methods. Russell Hilmoe remained my able and intelligent technician for many productive years; he was particularly good at adapting to new situations. Marie Lipsett, who had a good grasp of physical chemistry, joined the laboratory group; she collaborated with Dan Bradley on the study of complex formation between oligonucleotides and homopolymers. The flow of visitors continued as many people began to investigate nucleic acid enzymology. Littauer and I. R. (Bob) Lehman visited from Kornberg's department in St. Louis. Gobind Khorana's occasional visits were a joy as they gave me a chance to observe the development of his work and share in his good company as well as collaborate. Several times I also visited in Khorana's laboratory. Audrey Stevens was an especially brilliant postdoctoral fellow; all on her own she was one of the people who simultaneously discovered RNA polymerase. Altogether, it was an enjoyable and exciting time. After some years, however, I decided to turn to a different problem: the properties of bacterial membranes. Harold Neu, a medical postdoctoral fellow, joined me in the new investigations. The first problem he tackled was the location of ribonuclease in E. coli. At that time, a ribonuclease had been found associated with the 30 S ribosomes of the bacteria. Neu showed that the ribonuclease was actually in the periplasmic space between the cell membrane and the cell wall but binds to the 30 S ribosomes when the cell is split open (10Neu H.C. Heppel L.A. On the surface localization of enzymes in E. coli.Biochem. Biophys. Res. Commun. 1964; 17: 215Crossref PubMed Scopus (55) Google Scholar, 11Neu H.C. Heppel L.A. Some observations on the “latent” ribonuclease of Escherichia coli.Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 1267Crossref PubMed Scopus (33) Google Scholar). With special care, it was possible to obtain ribosomes free of ribonuclease. Thus, the ribonuclease is a periplasmic enzyme with no connection to ribosomes. In the course of this work, Nancy Nossal, a postdoctoral fellow, contributed to the development of Neu's procedure for the osmotic shock of the cells (12Neu H.C. Heppel L.A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts.J. Biol. Chem. 1965; 240: 3685Abstract Full Text PDF PubMed Google Scholar). The protocol made it possible to recover enzymes in high yield from the periplasmic space of Gram-negative bacteria. The procedure has since been used in many laboratories. Neu, and later others, discovered a number of other periplasmic enzymes, all located in the space between the cell membrane and cell wall. Anraku, a visitor from Japan, was very quiet but very effective and productive. He observed that Gram-negative bacteria able to transport d-galactose contain a specific periplasmic protein that can bind that sugar. A similar observation was made in the laboratory of Arthur Pardee. In the next few years, a large number of binding proteins were discovered in my laboratory and elsewhere. At NIH, several additional postdoctoral fellows contributed to this work. H. R. Dvorak, an M.D., had a special interest in metalloproteins. He and R. W. Brockman, a hard worker who visited the laboratory from Alabama, also worked on phosphatases released from E. coli by osmotic shock. In 1967, Efraim Racker induced me to join the Department of Biochemistry at Cornell University. The move was the beginning of more than 30 pleasant and productive years in Ithaca. The first postdoctoral fellow to join the laboratory, George Dietz, was an able and pleasant young man who studied the uptake of hexose phosphates by E. coli. Joel Weiner, a graduate student from Canada, and Clem Furlong, a postdoctoral fellow, worked on amino acid transport in E. coli including leucine-specific and glutamine-specific (13Weiner J.H. Heppel L.A. A binding protein for glutamine and its relation to active transport in Escherichia coli.J. Biol. Chem. 1971; 246: 6933Abstract Full Text PDF Google Scholar) periplasmic binding proteins. Furlong was an especially good experimentalist and was helpful with equipment problems. Weiner later became an outstanding member of the Canadian Biochemical Society. Ed Berger, a graduate student, carried out a landmark study showing that there are different mechanisms of energy coupling for the active transport of proline and glutamine in E. coli (14Berger E.A. Different mechanisms of energy coupling for the active transport of proline and glutamine in Escherichia coli.Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 1514Crossref PubMed Scopus (139) Google Scholar); this work received much favorable attention. Another member of the early group at Cornell was postdoctoral fellow Barry Rosen. He studied basic amino acid transport in E. coli, another process that involved a binding protein. Other students, postdoctoral fellows, and visitors contributed to our growing understanding of the periplasmic space and transport. Susan Curtis looked at the mechanism of ribose uptake, which involved energy from ATP rather than an energized membrane. James Cowell noted a similar result for glycylglycine. Janet Wood, a very able Canadian, worked on l-leucine transport. J. B. Smith and a graduate student, Paul Sternweis, purified the two “minor” subunits of F1-ATPase and examined their properties (15Smith J.B. Sternweis P.C. Purification of membrane attachment and inhibitory subunits of the proton translocating adenosine triphosphatase from E. coli.Biochem. J. 1977; 16: 306-311Crossref Scopus (135) Google Scholar). I was able to help Smith during a period when jobs were difficult to get and was delighted when he began doing independent work. T. Kitagawa made an interesting finding when he showed that the osmotic shock procedure does not necessarily kill the cells; some cells remain viable. Stanley Dunn and Masamitsu Futai used their time in the laboratory purifying and reconstituting the E. coli F1-ATPase (16Dunn S.D. Futai M. Reconstitution of a functional coupling factor from the isolated subunits of E. coli F1-ATPase.J. Biol. Chem. 1980; 255: 113-118Abstract Full Text PDF PubMed Google Scholar). Nizar Makan from India spent several postdoctoral years on exhaustive work that yielded evidence for metabolic processes that might be involved in permeabilization. In 1975, I decided to gain more experience in animal cell research. A half-year sabbatical was granted and I spent it with Henry Rozengurt in London. In the ensuing years, I made six additional visits of several months each to the Rozengurt laboratory. On one of these visits, I observed that 3T6 cells, which are spontaneously transformed, leaked nucleotides when 50 μm ATP is added to the medium; the effect is highly specific for ATP. Many excellent investigators have since studied this phenomenon, and G. Weisman, I. Friedberg, and I reviewed this work in 1986 (17Heppel L.A. Weisman G.A. Friedberg I. Permeabilization of transformed cells in culture by external ATP.J. Membr. Biol. 1986; 86: 189-196Crossref Scopus (60) Google Scholar). Friedberg received his degree for the work in my laboratory in about 1980. The most recent years in my laboratory included Ding-ji Wang and Ning-na Huang. They showed that ATP, in concentrations of a few micromolar, was a mitogen and explored this important effect of extracellular ATP in a series of papers (18Huang N. Wang D. Heppel L.A. Extracellular ATP is a mitogen for 3T3, 3T6 and A43l cells and acts synergistically with other growth factors.Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7904-7908Crossref PubMed Scopus (136) Google Scholar). I want also to mention a few other people who were in my laboratory at various times and whose collaboration I value. They include R. G. Alfonzo from Venezuela, K. Jacobson, a skilled organic chemist, and the productive Fernando Gonzalez, a graduate student and postdoctoral fellow. Barun De was a persistent and hard worker. Ahmed Ahmed came to the United States on a number of occasions to learn modern biology; he is a well known Professor of Plant Science and Toxicology in his native Egypt. I was also fortunate to know Gary Weisman and to watch with pleasure as he developed into a leader in his field. In the early 1980s I was able to spend 13 months (divided into short periods) back at NIH as a Fogarty Scholar in the laboratory of Claude Klee. It was good to be able to spend the entire day doing experiments at the bench. Klee is remarkable for being able to do experiments at the same time that she was running the Laboratory of Biochemistry in the National Cancer Institute. These reminiscences cover about 75 years. They are based on what I remember and no claims for accuracy are made. Selected references and reviews are included for the interested reader, and these sources also describe similar work done in other laboratories.