Happily at Work

It is a great privilege to be asked for a “Reflections” essay; I admire those prepared by my predecessors. My teachers were less prestigious than Arthur Kornberg's (1Kornberg A. Remembering our teachers..J. Biol. Chem. 2001; 276: 3-11Abstract Full Text Full Text PDF PubMed Google Scholar), and there was no single major theme in my research as was the case with several previous contributors to this series. Instead we studied a wide variety of metabolic phenomena that I have described in a summary of my first 50 years of biochemical research (2Lardy H.A. Semenza G. A half century of biochemistry. In A History of Biochemistry. Elsevier Science Publishing Co., Inc., New York1985: 297-325Google Scholar). Our findings included a treatment for selenium poisoning in livestock (undergraduate thesis; selenium-containing mercapturic acids are excreted in the urine) that was applied successfully to a human case; our studies of spermatozoa will be described in a following section. We elucidated the mechanism by which l-glyceraldehyde inhibits glycolysis (3Lardy H. Wiebelhaus V. Mann K. The mechanism by which glyceraldehyde inhibits glycolysis..J. Biol. Chem. 1950; 187: 325-337Abstract Full Text PDF PubMed Google Scholar). That disproved Needham's non-phosphorylating glycolysis in embryos and tumors. Could that have encouraged him to drop experiments and to devote his talents to prepare his magnificent history of Chinese science instead? We found that the function of biotin was to fix CO2 in heterotrophic organisms (4Lardy H. Potter R. Elvehjem C. The role of biotin in bicarbonate utilization by bacteria..J. Biol. Chem. 1947; 169: 451-452Abstract Full Text PDF PubMed Google Scholar); cellular respiration rates varied with the availability of inorganic P and phosphate acceptor (5Lardy H. The role of phosphate in metabolic control mechanisms..The Biology of Phosphorus. Michigan State College Press, East Lansing, MI1952: 287-294Google Scholar, 6Lardy H. Wellman H. Oxidative phosphorylations: role of inorganic phosphate and acceptor systems in control of metabolic rates..J. Biol. Chem. 1952; 195: 215-224Abstract Full Text PDF PubMed Google Scholar); propionate was metabolized by CO2 addition to ultimately yield succinate (7Lardy H. Peanasky R. Metabolic effects of biotin..Physiol. Rev. 1953; 33: 560-565Crossref PubMed Scopus (18) Google Scholar, 8Lardy H. Adler J. Synthesis of succinate from propionate and bicarbonate by soluble enzymes from liver mitochondria..J. Biol. Chem. 1956; 219: 933-942Abstract Full Text PDF PubMed Google Scholar). My students purified and crystallized some 10 phosphate-transferring enzymes, and we demonstrated that most of them required MgATP as substrate and were inhibited by free ATP; we found 16 different antibiotics that affected oxidative phosphorylation (9Lardy H. Reed P. Lin C.-H. Antibiotic inhibitors of mitochondrial ATP synthesis..Fed. Proc. 1975; 34: 1707-1710PubMed Google Scholar, 10Lardy H. Antibiotic inhibitors of mitochondrial energy transfer..Pharmacol. Ther. 1980; 11: 649-660Crossref PubMed Scopus (26) Google Scholar) and a dozen that acted as ionophores (11Lardy H. Graven S. Estrada-O S. Specific induction and inhibition of cation and anion transport in mitochondria..Fed. Proc. 1967; 26: 1355-1360PubMed Google Scholar), some of which are still being used in experiments. We also found that caffeine increased respiration and dramatically induced whiplash-type motility in sperm by increasing cyclic AMP (12Garbers D. Lust W. First N. Lardy H. Effects of phosphodiesterase inhibitors and cyclic nucleotides on sperm respiration and motility..Biochemistry. 1971; 10: 1825-1831Crossref Scopus (209) Google Scholar, 13Garbers D. First N. Lardy H. The stimulation of bovine sperm metabolism by cyclic nucleotide phosphodiesterase inhibitors..Biol. Reprod. 1973; 8: 589-598Crossref PubMed Scopus (50) Google Scholar); the respiratory response was dependent on the utilization of acetylcarnitine (14Milkowski A. Babcock D. Lardy H. Activation of bovine epididymal sperm respiration by caffeine..Arch. Biochem. Biophys. 1976; 176: 250-256Crossref PubMed Scopus (28) Google Scholar). Thyroid hormone and also dehydroepiandrosterone induced the synthesis of mitochondrial glycerol-3-phosphate dehydrogenase to as much as 20 times the normal concentration (15Lee Y.-P. Takemori A. Lardy H. Enhanced oxidation of α-glycerophosphate by mitochondria of thyroid-fed rats..J. Biol. Chem. 1959; 234: 3051-3054Abstract Full Text PDF PubMed Google Scholar, 16Lee Y.-P. Lardy H. Influence of thyroid hormones on l-α-glycerophosphate dehydrogenasees and other dehydrogenases in various organs of the rat..J. Biol. Chem. 1965; 240: 1427-1436Abstract Full Text PDF PubMed Google Scholar, 17Lardy H., Su, C.-Y. Kneer N. Wielgus S. Lardy H. Stratman F. Dehydroepiandrosterone induces enzymes that permit thermogenesis and decrease metabolic efficiency. In Hormones, Thermogenesis, and Obesity. Elsevier Science Publishers B.V., Amsterdam1989: 415-426Google Scholar) and formed part of a thermogenic system (17Lardy H., Su, C.-Y. Kneer N. Wielgus S. Lardy H. Stratman F. Dehydroepiandrosterone induces enzymes that permit thermogenesis and decrease metabolic efficiency. In Hormones, Thermogenesis, and Obesity. Elsevier Science Publishers B.V., Amsterdam1989: 415-426Google Scholar,18Lardy H. Watson R.R. Dehydroepiandrosterone and ergosteroids affect energy expenditure. In Health Promotion and Aging. Harwood, Amsterdam1999: 33-42Google Scholar). The path of carbon in gluconeogenesis was found to involve carboxylation of pyruvate (Utter reaction) in mitochondria, reduction of oxalacetate to malate, malate transport to cytosol in exchange for pyruvate, oxidation of malate to oxalacetate (the precursor of phosphopyruvate) together with the generation of the NADH required to reduce 3-phosphoglycerate to triose phosphate (19Lardy H. Paetkau V. Walter P. Paths of carbon in gluconeogenesis and lipogenesis: I. The role of mitochondria in supplying precursors of phosphoenolpyruvate..Proc. Natl. Acad. Sci. U. S. A. 1965; 53: 1410-1415Crossref PubMed Scopus (154) Google Scholar, 20Walter P. Paetkau V. Lardy H. Paths of carbon in gluconeogenesis and lipogenesis: III..J. Biol. Chem. 1966; 241: 2523-2532Abstract Full Text PDF PubMed Google Scholar); serine was found to be converted to glucose by an entirely different pathway, probably the reverse of its synthesis from hydroxypyruvate (21Lardy H. Veneziale C. Gabrielli F. Paths of carbon in gluconeogenesis..FEBS Symp. 1969; 19: 55-62Google Scholar). We also found that levels of liver cytosolic phosphoenolpyruvate carboxykinase (PEPCK) are regulated by the need for gluconeogenesis; they are increased by fasting and decreased in well fed animals; PEPCK is activated by ferrous ion, and in liver free calcium activates PEPCK by releasing Fe2+ from mitochondria to the cytosol (22Merryfield M. Lardy H. Ca2+-mediated activation of phosphoenolpyruvate carboxykinase occurs via the release of Fe2+ from rat liver mitochondria..J. Biol. Chem. 1982; 257: 3628-3635Abstract Full Text PDF PubMed Google Scholar); feeding tryptophan inhibits gluconeogenesis because its metabolite, quinolinate, forms a complex with ferrous ion that blocks PEPCK (23Veneziale C. Walter P. Kneer N. Lardy H. Influence of tryptophan and its metabolites on gluconeogenesis in the isolated, perfused liver..Biochemistry. 1967; 6: 2129-2138Crossref PubMed Scopus (147) Google Scholar,24Snoke R. Johnston J. Lardy H. Response of phosphopyruvate carboxylase to tryptophan metabolites and metal ions..Eur. J. Biochem. 1971; 24: 342-346Crossref PubMed Scopus (57) Google Scholar). The widely reported enhancement of liver mitochondrial respiration following exercise or the administration of glucagon or adrenaline to rats was found to be mediated by elevated malate concentration in the liver (25Bobyleva V. Lardy H. The role of malate in exercise-induced enhancement of mitochondrial respiration..Arch. Biochem. Biophys. 1986; 245: 470-476Crossref PubMed Scopus (19) Google Scholar, 26Bobyleva V. Wehbie R. Lardy H. The role of malate in hormone-induced enhancement of mitochondrial respiration..Arch. Biochem. Biophys. 1986; 245: 477-482Crossref PubMed Scopus (18) Google Scholar). Malate is known to facilitate mitochondrial uptake of substrates by exchange across the mitochondrial membranes. Naturally there was also an abundance of studies that yielded useful facts but not new concepts and many experiments undertaken to test hypotheses that turned out to be without merit! One reason for the diversity of research is that we wanted graduate students to have their own thesis research problems. Sixty-four candidates earned their Ph.D. degree in our group between 1945 and 1989 and more than 100 postdoctorate fellows conducted their research in our laboratories at the Institute for Enzyme Research. Relationships with these scholars and friends have always meant a great deal to me. At the time my research history was written (2Lardy H.A. Semenza G. A half century of biochemistry. In A History of Biochemistry. Elsevier Science Publishing Co., Inc., New York1985: 297-325Google Scholar), we were studying an intriguing class of Janus-like proteins, caltrins, that function in fertilization. Because the work was in progress it was not described in that essay. The caltrins have not been widely publicized and therefore are probably not familiar to most biochemists. The caltrins of different species have widely different structures and their multiple functions are achieved by disparate mechanisms. No aspect of living processes is more awe-inspiring than the union of a microscopic spermatozoon with an egg of the same species to initiate a new life. In this process the contribution of the male is to present a set of haploid chromosomes to join those of the egg. However, this presentation is a complex ceremony involving “capacitation,”i.e. alteration of sperm plasma membranes to permit penetration by Ca2+. Calcium uptake is followed by disruption of the a and that are converted to The sperm to the of the a and the enzymes the to a path for sperm Calcium uptake by the in the sperm the of motility by and of the sperm the sperm to in to the and the egg. The role of caltrins in of these processes is complex and Our work with spermatozoa been my and I a for the of sperm H.A. of Scholar). It the of motility and for and the in Because we the I was free to of sperm metabolism and the of energy for The findings that included the first the mechanism by which that decreased the motility of the the processes of glycolysis and oxidation are an of the mechanism with the that oxidation and glycolysis the energy is as than as H.A. The of and on sperm Biol. Chem. Full Text PDF Google Scholar). that same we most substrates that increased respiration and motility H.A. The metabolism of bovine epididymal Biochem. 6: Scholar, H.A. of and oxidation in Biochem. 6: Scholar), was in that it respiration but an degree of It is that the oxidation of this metabolite, in is more with phosphorylation than is the oxidation of the H.A. The metabolism of bovine epididymal Biochem. 6: Scholar). also to to the K. N. K. and mitochondrial energy J. PubMed Scopus Google Scholar) and have R. Lardy H.A. 2001; PubMed Scopus Google Scholar). a century the demonstrated that of not in the of J. the of the which or the of the the Google Scholar, J. and of Press, Scholar). is on the need for to the of the membranes on the sperm reaction) of on the of calcium Google Scholar) and First Lardy H.A. of the A at the cellular Biol. Chem. 1976; Full Text PDF PubMed Google Scholar, R. Calcium for in Reprod. 19: PubMed Scopus Google Scholar). We been the role of calcium transport in the of sperm the and enhancement of motility for some years First Lardy H.A. of the A at the cellular Biol. Chem. 1976; Full Text PDF PubMed Google Scholar, Babcock Lardy H.A. is a of of J. PubMed Scopus Google Scholar, Babcock Lardy H.A. of in Reprod. 1980; PubMed Scopus Google Scholar) Babcock found that the uptake of calcium by bovine epididymal sperm not in sperm from Lardy H.A. of to calcium of bovine Biol. PubMed Scopus Google Scholar). sperm of and will to 50 sperm in a calcium and an energy as Babcock Lardy H.A. is a of of J. PubMed Scopus Google Scholar, Lardy H.A. of to calcium of bovine Biol. PubMed Scopus Google Scholar, Babcock Lardy H.A. of in Reprod. 1980; PubMed Scopus Google Scholar). bovine sperm have the same calcium being in calcium in free of and in sperm still not this The was to to epididymal which demonstrated the of a calcium transport that we The was purified to Babcock Lardy H.A. and of a calcium transport from bovine Biol. Chem. 1982; 257: Full Text PDF PubMed Google Scholar) and the was J. W. Lardy H.A. The of the of bovine Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). A with the of was demonstrated to be to plasma membranes of sperm and was not on the membranes of epididymal sperm Lardy H.A. of calcium in bovine Biol. Chem. Full Text PDF PubMed Google Scholar). The also that a bovine been from bovine and was described as from bovine plasma which in by inhibition of PubMed Scopus Google Scholar). in the of R. of an from J. Google Scholar) were N. and are the same 1986; PubMed Scopus Google Scholar), our and the of and for bovine on inhibition of calcium uptake by epididymal sperm that as much as was required to calcium uptake by the sperm present in an as a of calcium transport and an of calcium uptake J. P. Lardy H.A. and function of the of J. PubMed Scopus Google Scholar). be by the to a cation The the and of calcium The of the that calcium transport inhibition to the the converted to a calcium transport and the stimulation of calcium uptake but not the to an J. Lardy H.A. plasma the of the of bovine Biol. Chem. Full Text PDF Google Scholar). the of were and from J. Lardy H.A. and of in the of the Reprod. PubMed Scopus Google Scholar, J. Lardy H.A. and of from of the Biol. Chem. Full Text PDF PubMed Google Scholar). were no in these caltrins I and and the with bovine was a group of the of bovine and I the these caltrins as with A J. Lardy H.A. and of in the of the Reprod. PubMed Scopus Google Scholar, J. Lardy H.A. and of from of the Biol. Chem. Full Text PDF PubMed Google Scholar); bovine not Babcock Lardy H.A. and of a calcium transport from bovine Biol. Chem. 1982; 257: Full Text PDF PubMed Google Scholar). The of the of caltrins I and are and The inhibition of calcium uptake sperm by of the caltrins of caltrins I and to the of calcium uptake by epididymal they caltrins Lardy H.A. of from of the Reprod. 33: PubMed Scopus (28) Google Scholar). The of rats and caltrins that have been purified and Lardy H.A. and of from of the rat and Biol. Chem. Full Text PDF PubMed Google Scholar). are and is from a precursor in the Lardy H. and of a precursor of the calcium transport from of the Reprod. PubMed Scopus Google Scholar); the of rat and its precursor is C. A. of calcium transport of PubMed Scopus Google Scholar). The rat a of acids with a of I. rat caltrins have to or bovine of the caltrins, with the of the that are not with the been with as the of rat and the but not the on calcium The of the are known A. of rat and and Reprod. PubMed Scopus Google Scholar). In the case of which the to be a formed between the the to also as a of by and but from its we found a of and no A been purified from by and Lin A in as a sperm motility Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). It of which are with our for Lardy H.A. and of from of the rat and Biol. Chem. Full Text PDF PubMed Google Scholar). Lin A in as a sperm motility Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) the of their from the and the first acids by they the to in our Our was by the applied to different from to of the the acids in of these the same we are of our and that there are between and of the be by single The group Lin of a in and of its on sperm Reprod. PubMed Scopus Google Scholar) reported from with no to our the and the of sperm but not to the part of the to the which the mitochondria J. P. Lardy H.A. and function of the of J. PubMed Scopus Google Scholar). sperm from the same as epididymal sperm with but epididymal sperm not to not the J. P. Lardy H.A. and function of the of J. PubMed Scopus Google Scholar). at these was to the and the of functions were in the case of sperm The test I to the sperm the but not to other of the sperm I inhibited release from the of in the of many of these sperm were but Lardy H.A. of from of the Reprod. 33: PubMed Scopus (28) Google Scholar). epididymal sperm not with caltrins and were calcium to the and the to the sperm and to the It release from the and of the sperm motility because calcium to the mechanism was J. Lardy H.A. and of in the of the Reprod. PubMed Scopus Google J. Lardy H.A. and of from of the Biol. Chem. Full Text PDF PubMed Google Scholar). of and function for the that of the caltrins inhibits of the calcium uptake that occurs in the of these Lardy H.A. of from of the Reprod. 33: PubMed Scopus (28) Google of to epididymal are the same as described in the to that sperm were with purified and the been prepared with to the sperm with from Lardy H.A. of from of the Reprod. 33: PubMed Scopus (28) Google The D. of plasma on and in human Reprod. PubMed Scopus Google Scholar) that of in the of plasma on and in the present for the of in the in for the human sperm been demonstrated much in sperm Lardy H.A. of from of the Reprod. 33: PubMed Scopus (28) Google Scholar). years H. S. R. M. H. V. Scholar) reported the of in and their been by several and I are also inhibitors A. of rat and and Reprod. PubMed Scopus Google Scholar), and the is with the inhibitors from the by K. M. A. N. N. S. F. and of inhibitors in rat Chem. Google Scholar) and from liver by H. N. A from rat is to rat J. Biochem. 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In the role of caltrins in be from their known spermatozoa caltrins as well as other caltrins calcium the and a The and enzymes are calcium uptake by the caltrins the sperm some time the sperm will have the and the the will have been from the in the case of bovine and the in the case of The forms of calcium uptake at the it activates and at the it induces of the sperm that caltrins have that at the and and the other to the sperm and of these in our was for of on my in but is being by in The of its more than Press, New Scholar); I was to a for but not for metabolic A that was hormones from dehydroepiandrosterone was known as an in the of to and in it and in J. PubMed Scopus Google Scholar), and decreased in R. of on the effects of dehydroepiandrosterone in and in normal 33: PubMed Google Scholar), decreased the of and in of and in by treatment with PubMed Scopus Google Scholar), R. A. W. with the 26: PubMed Scopus Google Scholar), and in effects in male of and from Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). some that was converted to more that these in the many studied the of to other by and but a of the were for We a of of that were metabolites in the of or more new a an for and our research thyroid hormone to rats induces the of mitochondrial dehydrogenase to 20 times the normal in liver (15Lee Y.-P. Takemori A. Lardy H. Enhanced oxidation of α-glycerophosphate by mitochondria of thyroid-fed rats..J. Biol. Chem. 1959; 234: 3051-3054Abstract Full Text PDF PubMed Google Scholar, 16Lee Y.-P. Lardy H. Influence of thyroid hormones on l-α-glycerophosphate dehydrogenasees and other dehydrogenases in various organs of the rat..J. Biol. Chem. 1965; 240: 1427-1436Abstract Full Text PDF PubMed Google Scholar) and less in other Y.-P. Lardy H. Influence of thyroid hormones on l-α-glycerophosphate dehydrogenasees and other dehydrogenases in various organs of the rat..J. Biol. Chem. 1965; 240: 1427-1436Abstract Full Text PDF PubMed Google Scholar). A. S. N. Effects of dehydroepiandrosterone on and of male and rats..J. 1986; PubMed Scopus Google Scholar) reported that metabolism and we found this induced the of but not that of other (17Lardy H., Su, C.-Y. Kneer N. Wielgus S. Lardy H. Stratman F. Dehydroepiandrosterone induces enzymes that permit thermogenesis and decrease metabolic efficiency. In Hormones, Thermogenesis, and Obesity. Elsevier Science Publishers B.V., Amsterdam1989: 415-426Google Scholar, C.-Y. Lardy H. of mitochondrial dehydrogenase in rats by Biochem. PubMed Scopus Google Scholar). is also increased by these hormones H. S. J. of dehydroepiandrosterone and on liver enzymes and J. PubMed Scopus Google Scholar), and the response of these enzymes to a of The enzymes a thermogenic system regulated by calcium and other (17Lardy H., Su, C.-Y. Kneer N. Wielgus S. Lardy H. Stratman F. Dehydroepiandrosterone induces enzymes that permit thermogenesis and decrease metabolic efficiency. In Hormones, Thermogenesis, and Obesity. Elsevier Science Publishers B.V., Amsterdam1989: 415-426Google Scholar, H. Watson R.R. Dehydroepiandrosterone and ergosteroids affect energy expenditure. In Health Promotion and Aging. Harwood, Amsterdam1999: 33-42Google Scholar, M. Lardy H. stimulation of rat liver mitochondrial Biol. Chem. Full Text PDF PubMed Google Scholar, H. Kneer N. M. V. of thermogenic enzymes by and its N. Acad. Sci. PubMed Scopus Google Scholar). We found that of at other than the to the thermogenic enzymes H. Kneer N. P. metabolites and of PubMed Scopus Google Scholar). and were more than Because increased in that we that the same was in to an with liver with and malate and the at time that was A. P. Lardy H. metabolism of dehydroepiandrosterone by rat liver in a Scopus Google Scholar) and is in The and of the many formed from by liver were because of the of A. P. Lardy H. and of a for the of in human Scopus Google Scholar, A. P. Lardy H. of in rat plasma ion 2001; Scopus Google Scholar, A. P. Lardy H. of A. 2001; PubMed Scopus (18) Google Scholar, A. P. Lardy H. induced of and their a Chem. PubMed Scopus Google Scholar, A. P. Lardy H. of response of in by A. PubMed Scopus Google Scholar). to be some or at not as precursors of or and therefore are no in rats H. S. C. of in Biophys. PubMed Scopus Google Scholar) or S. C. Lardy H. of in Biophys. PubMed Scopus Google Scholar) in and in a I was well by normal to for M. A. R. K. P. C. Lardy H. and with of in male Scholar). at that for in of were to more and than those D. C. M. G. A of in Ther. Scholar). was more than as an of in and in in with J. S. Lardy H. The of on in and Scopus Google Scholar). of the of metabolism in been known for many years to have It also activates in H. S. Lardy H. C. is a hormone with in human Natl. Acad. Sci. U. S. A. 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