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The Kennedy Pathway for Phospholipid Synthesis: the Work of Eugene Kennedy

2005; Elsevier BV; Volume: 280; Issue: 25 Linguagem: Inglês

10.1016/s0021-9258(20)67490-7

1083-351X

Nicole Kresge, Robert Simoni, Robert L. Hill,

Metabolism and Genetic Disorders

Oxidation of Fatty Acids and Tricarboxylic Acid Cycle Intermediates by Isolated Rat Liver Mitochondria (Kennedy, E. P., and Lehninger, A. L. (1949) J. Biol. Chem. 179, 957–972) The Function of Cytidine Coenzymes in the Biosynthesis of Phospholipides (Kennedy, E. P., and Weiss, S. B. (1956) J. Biol. Chem. 222, 193–214) Eugene Patrick Kennedy was born in Chicago in 1919. He enrolled at De Paul University in 1937 as a chemistry major and then went to the University of Chicago in 1941 for graduate training in organic chemistry. To pay his tuition, Kennedy also got a job in the chemical research department of Armour and Company, one of the large meat packers in Chicago. As part of the war effort, his job at Armour was to assist in the large scale fractionation of bovine blood to obtain pure bovine serum albumin. It was believed that the bovine serum albumin might be useful for treating shock in soldiers on the battlefield. However, by the end of 1942, hope had faded that bovine serum albumin would be an effective treatment, and the Red Cross started to collect blood from volunteers instead. Armour opened a new facility in Fort Worth, Texas for the fractionation of human blood from donors, and Kennedy was sent to Fort Worth to assist in this effort. He remained in Texas until 1945, when the war was clearly nearing its end and large amounts of human plasma proteins had been stockpiled. Returning to the University of Chicago, Kennedy immediately transferred from the Department of Chemistry to the Department of Biochemistry. His experience on the plasma project had led to a new appreciation of biochemistry. When he was ready to begin research for his dissertation, Kennedy approached Albert Lehninger, a young faculty member whose earlier research was the subject of a previous Journal of Biological Chemistry (JBC) Classic (1JBC Classic Lehninger A.L. J. Biol. Chem. 1945; 157 (http://www.jbc.org/cgi/content/full/280/14/e11): 363-382Abstract Full Text PDF Google Scholar). At that time, Lehninger was studying oxidative phosphorylation and fatty acid oxidation. Kennedy writes, “With staggering naiveté, I suggested to him that the proper approach would be to purify the various enzymes undoubtedly involved in fatty acid oxidation and crystallize them. He agreed that this would be desirable, but went on to point out rather gently that fatty acid oxidation had not yet been demonstrated in a soluble extract from which individual enzymes might be isolated. To reach that stage, it would first be necessary to discover the nature of the energy-requiring activation or”sparking“of fatty acid oxidation and the special dependence of the process on particulate structures” (2Kennedy E. Sailing to Byzantium..Annu. Rev. Biochem. 1992; 61: 1-28Crossref PubMed Scopus (23) Google Scholar). Despite this initial incident, Lehninger agreed to take Kennedy on as a graduate student, and he began to work on the problem of fatty acid oxidation in 1947. Lehninger had observed that both fatty acid oxidation and oxidative phosphorylation were inhibited in a strikingly parallel fashion when particulate enzyme preparations of homogenized rat livers were exposed to hypotonic buffers. The activity could be preserved by adding either salts or iso-osmotic amounts of sucrose to the buffers. Kennedy's first project in the laboratory was a detailed study of these effects (3Lehninger A.L Kennedy E.P. The requirements of the fatty acid oxidase complex of rat liver..J. Biol. Chem. 1948; 173: 753-771Abstract Full Text PDF PubMed Google Scholar). These studies led Lehninger and Kennedy to surmise that fatty acid oxidation, oxidative phosphorylation, and the Krebs cycle must all be taking place in one organelle, bounded by a membrane impermeable to certain solutes. Although their enzyme preparations were quite crude, they were convinced that the organelle was the mitochondrion, even though functionally and morphologically intact mitochondria had not yet been isolated. Around this time George Palade and his collaborators were developing methods for the separation and identification of organelles. As reported in a previous JBC Classic (4JBC Classic Hogeboom G.H. Schneider W.C. Palade G.E. J. Biol. Chem. 1948; 172 (http://www.jbc.org/cgi/content/full/280/22/e19): 619-635Abstract Full Text PDF PubMed Google Scholar), Palade worked out a method for the isolation of purified mitochondria by differential centrifugation in 0.88 m sucrose. Kennedy immediately tested mitochondria isolated by this method and obtained convincing evidence that oxidative phosphorylation, fatty acid oxidation, and the reactions of the Krebs cycle did indeed occur in the mitochondria. This is the subject of the first JBC Classic reprinted here. After finishing graduate school, Kennedy went to the University of California, Berkeley, to work with Horace A. Barker. Barker and his graduate student Earl Stadtman, both of whom will be featured in future JBC Classics, had just discovered that soluble extracts of Clostridium kluyveri cells could produce short-chain fatty acids from ethyl alcohol. Although the initial discovery had already been made, there was much to be learned about these extracts and Kennedy aided in this effort. In 1950, Kennedy joined Fritz Lipmann, author of a previous JBC Classic (5JBC Classic Lipmann F. J. Biol. Chem. 1945; 160 (http://www.jbc.org/cgi/content/full/280/21/e18): 173-190Abstract Full Text PDF Google Scholar), at Harvard Medical School. 1Please see Ref. 8Kennedy E.P. Hitler's gift and the era of biosynthesis..J. Biol. Chem. 2001; 276: 42619-42631Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar for Kennedy's JBC Reflection on Fritz Lipmann, Rudolf Schoenheimer, and Konrad Bloch. He then returned to the University of Chicago in 1951, after being given a joint appointment in the Department of Biochemistry and the newly organized Ben May Laboratory for Cancer Research. In Chicago, Kennedy started to study the origins of the phosphodiester bond of phosphatidylcholine using labeled choline. He found that free choline, but not phosphocholine, was converted to lipid in a reaction dependent on ATP generated by oxidative phosphorylation (6Kennedy E.P. The synthesis of lecithin in isolated mitochondria..J. Am. Chem. Soc. 1953; 75: 249-250Crossref Scopus (2) Google Scholar). At the same time, Kornberg and Pricer (7Kornberg A. Pricer W.E. Fed. Proc. 1952; 11: 242Google Scholar) reported experiments in which phosphocholine was converted to a lipid (later identified as lecithin) in a reaction that required ATP. Determined to understand why he and Kornberg had obtained contradicting results, Kennedy, along with his graduate student Samuel Weiss, undertook a detailed examination of the differences between the two studies. They discovered that they could reproduce Kornberg's results using commercially available ATP. However, large amounts of ATP were needed, suggesting that an impurity, rather than ATP, might be involved in the reaction. Kennedy and Weiss' discovery of the cofactor involved in the conversion of phosphocholine to lecithin is the subject of the second JBC Classic reprinted here. After testing several nucleoside triphosphates, they realized that cytidine triphosphate (CTP) was the active cofactor in the phosphocholine reaction. They formulated a number of schemes to account for the involvement of CTP in phospholipid synthesis and eventually decided that intermediary formation of cytidine diphosphate choline (CDP-choline) was occurring in the reaction. Although they had no evidence for its involvement, they synthesized CDP-choline and cytidine diphosphate ethanolamine and tested their abilities to act as cofactors in lipid biosynthesis. Using 14C to label the cytidine coenzymes, Kennedy and Weiss proved that CDP-choline and cytidine diphosphate ethanolamine were activated forms of phosphorylcholine and phosphorylethanolamine and were precursors of lecithin and phosphatidylethanolamine. They also showed that the two cytidine coenzymes were present in high quantities in liver and yeast. In 1959, Kennedy was invited to become a Hamilton Kuhn Professor and head of the Department of Biological Chemistry at the Harvard Medical School. He continued his research on phospholipid biosynthesis and was able to formulate a detailed picture of the pathways of biosynthesis of the principal glycerophosphatides and of triacylglycerol by 1961. Kennedy's interests also led him to investigate membrane biogenesis and function in bacteria, the translocation of membrane phospholipids, and periplasmic glucans and cell signaling in bacteria. Kennedy is currently at Harvard as the Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology, Emeritus. 2All biographical information on Eugene P. Kennedy was taken from Ref. 2Kennedy E. Sailing to Byzantium..Annu. Rev. Biochem. 1992; 61: 1-28Crossref PubMed Scopus (23) Google Scholar.