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• W2093025897 abstract "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 acrosome, a sac containing hyaluronidase and proproteinases that autocatalytically are converted to active acrosins. The sperm attach to the protective layer of the egg, a glycoprotein matrix (zona pellucida), and the calcium-activated acrosomal enzymes attack the zona to provide a path for sperm entry. Calcium uptake by the contractile components in the sperm tail facilitates the acquisition of “hyperactivated” motility characterized by rapid lashing and wider excursion of the sperm tail. This causes the sperm to swim in tight arcs to drive through the zona and then penetrate the egg. The role of caltrins in regulating each of these processes is complex and fascinating. Our work with spermatozoa had been continuous since 1939 when my professor, Paul Phillips, and I developed a medium for the preservation of animal sperm (27Lardy H.A. Phillips P.H. Preservation of spermatozoa..Proc. Am. Soc. Animal Production. 1939; 32: 219-221Google Scholar). It permitted the retention of motility and fertility for 8–12 days and launched the artificial insemination industry in livestock. Because we had solved the practical problem, I was free to study basic aspects of sperm metabolism and the regulation of energy capture for motility. The findings during that period included the first clear statement concerning the mechanism by which 2,4-dinitrophenol functions: “the fact that DNP decreased the motility of the spermatozoa, while the processes of glycolysis and oxidation are increased, indicates an interference of the energy-coupling mechanism with the result that oxidation and glycolysis run rampant, while the energy is lost as heat rather than as work” (28Lardy H.A. Phillips P.H. The effect of thyroxine and dinitrophenol on sperm metabolism..J. Biol. Chem. 1943; 149: 177-182Abstract Full Text PDF Google Scholar). During that same period we discovered that, unlike most substrates that increased both respiration and motility (29Lardy H.A. Hansen R.G. Phillips P.H. The metabolism of bovine epididymal spermatozoa..Arch. Biochem. 1945; 6: 41-51Google Scholar, 30Lardy H.A. Phillips P.H. Studies of fat and carbohydrate oxidation in mammalian spermatozoa..Arch. Biochem. 1945; 6: 53-61Google Scholar), “β-hydroxybutyrate was unusual in that it depressed endogenous respiration slightly, but supported an excellent degree of motility. It is possible that the oxidation of this metabolite, in spermatozoa, is more efficiently coupled with phosphorylation than is the oxidation of the endogenous lipid reserve” (29Lardy H.A. Hansen R.G. Phillips P.H. The metabolism of bovine epididymal spermatozoa..Arch. Biochem. 1945; 6: 41-51Google Scholar). This explanation seems also to apply to the working heart (31Sato K. Kashiwaya Y. Keon C.A. Tsuchiya N. King M.T. Radda G.K. Chance B. Clarke K. Veech R.L. Insulin, ketone bodies and mitochondrial energy transduction..FASEB J. 1995; 9: 651-658Crossref PubMed Scopus (313) Google Scholar) and may have therapeutic implications (32Veech R. Chance B. Kashiwaya Y. Lardy H.A. Cahill G.F. Ketone bodies, potential therapeutic uses..IUBMB Life. 2001; 51: 241-247Crossref PubMed Scopus (315) Google Scholar). Nearly a century ago the eminent physiologist Jacques Loeb demonstrated that fertilization of sea urchin eggs does not occur in the absence of Ca2+ (33Loeb J. On the nature of the conditions which determine or prevent the entrance of the spermatozoan into the egg..Am. Natur. 1915; 49: 257-285Crossref Google Scholar, 90Loeb J. Artificial Parthenogenesis and Fertilization. University of Chicago Press, Chicago, IL1913Google Scholar). This failure is based on the need for Ca2+ to promote the lysis of the acrosomal membranes on the sperm head (acrosomal reaction) of both invertebrates (34Dan J.C. Studies on the acrosome. III. Effect of calcium deficiency..Biol. Bull. 1954; 107: 335-349Crossref Google Scholar) and vertebrates (35Babcock D.F. First N.L. Lardy H.A. Action of the ionophore A 23187 at the cellular level..J. Biol. Chem. 1976; 251: 3881-3886Abstract Full Text PDF PubMed Google Scholar, 36Yanagimachi R. Calcium requirement for sperm-egg fusion in mammals..Biol. Reprod. 1978; 19: 949-958Crossref PubMed Scopus (106) Google Scholar). We had been investigating the role of calcium transport in the regulation of sperm behavior, including the acrosome reaction and enhancement of motility for some years (35Babcock D.F. First N.L. Lardy H.A. Action of the ionophore A 23187 at the cellular level..J. Biol. Chem. 1976; 251: 3881-3886Abstract Full Text PDF PubMed Google Scholar, 37Singh J.P. Babcock D.F. Lardy H.A. Increased calcium-ion influx is a component of capacitation of spermatozoa..Biochem. J. 1978; 172: 549-556Crossref PubMed Scopus (143) Google Scholar, 91Singh J.P. Babcock D.F. Lardy H.A. Induction of accelerated acrosome reaction in guinea pig sperm..Biol. Reprod. 1980; 22: 566-570Crossref PubMed Scopus (22) Google Scholar) when Donner Babcock found that the rapid uptake of calcium by bovine epididymal sperm did not occur in sperm separated from ejaculates (38Babcock D.E. Singh J.P. Lardy H.A. Alteration of membrane permeability to calcium ions during maturation of bovine spermatozoa..Dev. Biol. 1979; 69: 85-93Crossref PubMed Scopus (76) Google Scholar). Epididymal sperm contain 6 ± 1 nmol of calcium/108 cells and will accumulate up to 50 nmol/108 sperm when incubated in a medium containing 0.2 mm calcium and an oxidizable energy source such as β-hydroxybutyrate (37Singh J.P. Babcock D.F. Lardy H.A. Increased calcium-ion influx is a component of capacitation of spermatozoa..Biochem. J. 1978; 172: 549-556Crossref PubMed Scopus (143) Google Scholar, 38Babcock D.E. Singh J.P. Lardy H.A. Alteration of membrane permeability to calcium ions during maturation of bovine spermatozoa..Dev. Biol. 1979; 69: 85-93Crossref PubMed Scopus (76) Google Scholar, 91Singh J.P. Babcock D.F. Lardy H.A. Induction of accelerated acrosome reaction in guinea pig sperm..Biol. Reprod. 1980; 22: 566-570Crossref PubMed Scopus (22) Google Scholar). Ejaculated bovine sperm have the same low calcium content despite being bathed in 9 mm calcium in seminal fluid. When washed free of seminal fluid and suspended in media containing calcium, ejaculated sperm still do not take up this divalent cation. The obvious next experiment was to add seminal fluid to epididymal sperm, which demonstrated the presence of a calcium transport inhibitor that we termed caltrin. The inhibitor was purified to homogeneity (39Rufo G.A. Singh J.B. Babcock D.E. Lardy H.A. Purification and characterization of a calcium transport inhibitor protein from bovine seminal plasma..J. Biol. Chem. 1982; 257: 4627-4632Abstract Full Text PDF PubMed Google Scholar) and the amino acid sequence was determined (Fig. 1) (40Lewis R.V. San Agustin J. Kruggel W. Lardy H.A. The structure of caltrin, the calcium-transport inhibitor of bovine seminal plasma..Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6490-6491Crossref PubMed Scopus (42) Google Scholar). A protein with the properties of caltrin was demonstrated to be bound to plasma membranes of ejaculated sperm and was not detected on the membranes of epididymal sperm (41Rufo G.A. Schoff P.K. Lardy H.A. Regulation of calcium content in bovine spermatozoa..J. Biol. Chem. 1984; 259: 2547-2552Abstract Full Text PDF PubMed Google Scholar). The sequence also disclosed that a similar protein, termed bovine seminal plasmin, had been isolated from bovine semen and was described as having antimicrobial activity (42Reddy E.S.P. Bhargava P.M. Seminal plasmin—an antimicrobial protein from bovine seminal plasma which acts in E. coli by specific inhibition of rRNA synthesis..Nature. 1979; 279: 725-728Crossref PubMed Scopus (112) Google Scholar). Errors in the sequencing of seminal plasmin (43Theil R. Scheit K.H. Amino acid sequence of seminal plasmin, an antimicrobial protein from bull semen..EMBO J. 1983; 12: 1159-1163Crossref Google Scholar) were later corrected (44Sitaram N. Kumari V.K. Bhargava P.M. Seminal plasmin and caltrin are the same protein..FEBS Lett. 1986; 201: 233-236Crossref PubMed Scopus (45) Google Scholar), thus confirming our structure and the identity of seminal plasmin and caltrin. Analyses for bovine caltrin based on inhibition of calcium uptake by epididymal bull sperm indicated that bull seminal fluid contained about twice as much caltrin as was required to inhibit calcium uptake 90% by the sperm present in an ejaculate. On storage, caltrin lost activity as a blocker of calcium transport and became an enhancer of calcium uptake (45San Agustin J. Hughes P. Lardy H.A. Properties and function of caltrin, the calcium-transport inhibitor of bull seminal plasma..FASEB J. 1987; 1: 60-66Crossref PubMed Scopus (27) Google Scholar). This transformation could be accomplished rapidly by binding the fresh inhibitory caltrin to a cation exchanger. The recovered caltrinincreased both the rate and extent of calcium uptake. The acidic components of the eluate contained ether-extractable phospholipids that restored calcium transport inhibition to the enhancer caltrin protein. Among the pure phospholipids tested only phosphatidylserine converted enhancer caltrin to a calcium transport inhibitor. Phosphatidylcholine, phosphatidylinositol, and citrate abolished the stimulation of calcium uptake but did not change the enhancer to an inhibitor (46San Agustin J. Lardy H.A. Bovine seminal plasma constituents modulate the activity of caltrin, the calcium-transport regulating protein of bovine spermatozoa..J. Biol. Chem. 1990; 265: 6860-6867Abstract Full Text PDF Google Scholar). With the aid of anti-bovine caltrin antiserum, two caltrin proteins were detected and isolated from guinea pig seminal vesicle secretions (47Coronel C.E. San Agustin J. Lardy H.A. Identification and partial characterization of caltrin-like proteins in the reproductive tract of the guinea pig..Biol. Reprod. 1988; 38: 713-722Crossref PubMed Scopus (16) Google Scholar, 92Coronel C.E. San Agustin J. Lardy H.A. Purification and structure of caltrin-like proteins from seminal vesicle of the guinea pig..J. Biol. Chem. 1990; 265: 6854-6859Abstract Full Text PDF PubMed Google Scholar). There were no common amino acid sequences in these peptides designated G.P. caltrins I and II, and the only identity with bovine caltrin was a group of four (Gly-Asn-Arg-Ser) near the carboxyl terminus of bovine caltrin and G.P. caltrin I (Fig. 1); nonetheless, the anti-bovine caltrin antiserum recognizes these guinea pig proteins. Both G.P. caltrins contain carbohydrate residues as detected with concanavalin A (47Coronel C.E. San Agustin J. Lardy H.A. Identification and partial characterization of caltrin-like proteins in the reproductive tract of the guinea pig..Biol. Reprod. 1988; 38: 713-722Crossref PubMed Scopus (16) Google Scholar, 92Coronel C.E. San Agustin J. Lardy H.A. Purification and structure of caltrin-like proteins from seminal vesicle of the guinea pig..J. Biol. Chem. 1990; 265: 6854-6859Abstract Full Text PDF PubMed Google Scholar); bovine caltrin does not (39Rufo G.A. Singh J.B. Babcock D.E. Lardy H.A. Purification and characterization of a calcium transport inhibitor protein from bovine seminal plasma..J. Biol. Chem. 1982; 257: 4627-4632Abstract Full Text PDF PubMed Google Scholar). The molecular weights of the peptide portion of G.P. caltrins I and II are 5082 and 6255, respectively. The maximal inhibition of calcium uptake into guinea pig sperm by each of the G.P. caltrins approached 50%. Deglycosylation of G.P. caltrins using trifluoromethanesulfonic acid caused both I and II to enhance the rate of calcium uptake by guinea pig epididymal sperm, i.e. they become enhancer caltrins (48Coronel C.E. Lardy H.A. Functional properties of caltrin proteins from seminal vesicles of the guinea pig..Mol. Reprod. Dev. 1992; 33: 74-80Crossref PubMed Scopus (28) Google Scholar). The seminal vesicles of rats and mice contain caltrins that have been purified and sequenced (Fig. 1) (49Coronel C.E. Winnica D.E. Novella M.L. Lardy H.A. Purification, structure, and characterization of caltrin proteins from seminal vesicle of the rat and mouse..J. Biol. Chem. 1992; 267: 20909-20915Abstract Full Text PDF PubMed Google Scholar). Their calculated molecular weights are 6217 and 8476, respectively. Rat caltrin is derived from a 54-kDa inactive precursor produced in the seminal vesicles (50Coronel C.E. Novella M.L. Winnica D.E. Lardy H. Isolation and characterization of a 54-kilodalton precursor of caltrin, the calcium transport inhibitor protein from seminal vesicles of the rat..Biol. Reprod. 1993; 48: 1326-1333Crossref PubMed Scopus (9) Google Scholar); the biosynthesis of rat caltrin and its precursor is androgen-dependent (51Novella M.L. Maldonado C. Aoki A. Coronel C.E. Androgen-dependent synthesis/secretion of caltrin, calcium transport inhibitor protein of mammalian seminal vesicle..Arch. Androl. 1999; 42: 1-12Crossref PubMed Scopus (7) Google Scholar). The active rat protein has a sequence of 13 amino acids nearly identical with a segment of G.P. caltrin I. Neither rat nor mouse caltrins have any significant sequence similarity to G.P. caltrin II or bovine caltrin. Each of the caltrins, with the exception of the bovine, contains cysteine residues that are not reactive with thiol reagents until the protein has been treated with reducing agents such as dithiothreitol. Reducing the cystine disulfide bonds of rat caltrin and carboxymethylating the protein diminishes, but does not eliminate, the effect on calcium transport. The locations of the disulfide bonds are known (52Winnica D.E. Novella M.L. Dematteis A. Coronel C.E. Trypsin/acrosin inhibitor activity of rat and guinea pig caltrin proteins. Structural and functional studies..Biol. Reprod. 2000; 63: 42-48Crossref PubMed Scopus (20) Google Scholar). In the case of mouse caltrin, which contains 7 cysteine residues, the protein appears to be a disulfide dimer formed between the odd cysteines. Reduction converts the 17-kDa native mouse caltrin to 8.5 kDa. Bovine caltrin also behaves as a dimer of M r 9600–10,500 by gel permeation and gel electrophoresis, but from its amino acid content we found a M r of 5411 and no cysteine. A caltrin protein designated SVS VII has been purified from mouse seminal vesicles by Yee-Hsiung Chen and co-workers (53Luo C-W. Lin H-J. Chen Y-H. A novel heat-labile phospholipid-binding protein, SVS VII, in mouse seminal vesicle as a sperm motility enhancer..J. Biol. Chem. 2001; 276: 6913-6921Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). It has 76 amino acids, 71 of which are identical with our sequence for mouse caltrin (49Coronel C.E. Winnica D.E. Novella M.L. Lardy H.A. Purification, structure, and characterization of caltrin proteins from seminal vesicle of the rat and mouse..J. Biol. Chem. 1992; 267: 20909-20915Abstract Full Text PDF PubMed Google Scholar). Luo et al. (53Luo C-W. Lin H-J. Chen Y-H. A novel heat-labile phospholipid-binding protein, SVS VII, in mouse seminal vesicle as a sperm motility enhancer..J. Biol. Chem. 2001; 276: 6913-6921Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) determined the sequence of their protein from the corresponding cDNA and the first 18 amino acids by automated Edman degradation; they ascribe the differences to errors in our structure. Our sequence was determined by the Edman procedure applied to five different peptides isolated from caltrin subjected to partial proteolysis. Four of the peptides contained the amino acids in question; each of these fragments contained the same sequence. Therefore we are confident of our structural assignment and assume that there are genetic differences between CD-1 and Swiss white mice. Three of the differences can be explained by single base changes. The Chen group (54Chen L.-Y. Lin Y.-H. Lai M.-L. Chen Y.-H. Developmental profile of a caltrin-like protease inhibitor, P12, in mouse seminal vesicle and characterization of its binding sites on sperm surface..Biol. Reprod. 1998; 59: 1498-1505Crossref PubMed Scopus (46) Google Scholar) reported another caltrin-like protein, P12, from mouse seminal vesicles with no appreciable sequence similarity to our mouse caltrin. Bovine caltrin binds over the acrosome and the entire tail of bull sperm but does not bind to the posterior part of the head nor to the midpiece, which contains the mitochondria (45San Agustin J. Hughes P. Lardy H.A. Properties and function of caltrin, the calcium-transport inhibitor of bull seminal plasma..FASEB J. 1987; 1: 60-66Crossref PubMed Scopus (27) Google Scholar). Washed sperm separated from bull semen show the same fluorescence staining pattern as epididymal sperm treated with caltrin (Fig. 2), but epididymal sperm not exposed to caltrin do not bind the fluorescence-labeled antibody (45San Agustin J. Hughes P. Lardy H.A. Properties and function of caltrin, the calcium-transport inhibitor of bull seminal plasma..FASEB J. 1987; 1: 60-66Crossref PubMed Scopus (27) Google Scholar). Caltrin binding at these two sites was assumed to regulate the acrosome reaction and the hyperactivation of motility, respectively. These separate functions were clearly defined in the case of guinea pig sperm (see below). The immunofluorescence test showed G.P. caltrin I binding to the G.P. sperm over the acrosome but not to other parts of the sperm (Fig. 3). Caltrin I (0.5 mg/108sperm) nearly completely inhibited hyaluronidase release from the acrosome during 30 min of incubation in the presence of 1 mm calcium; many of these sperm were hyperactive but had intact acrosomes (48Coronel C.E. Lardy H.A. Functional properties of caltrin proteins from seminal vesicles of the guinea pig..Mol. Reprod. Dev. 1992; 33: 74-80Crossref PubMed Scopus (28) Google Scholar). Guinea pig epididymal sperm not treated with caltrins released hyaluronidase and were hyperactive, indicating calcium access to both the acrosome and the tail. Guinea pig caltrin II (0.5 mg/108 sperm) bound to the sperm tail and very sparingly to the head (Fig. 4). It depressed hyaluronidase release from the acrosome only partially, and during 2 h of incubation the sperm maintained linear motility because calcium access to the contractile mechanism was blocked (47Coronel C.E. San Agustin J. Lardy H.A. Identification and partial characterization of caltrin-like proteins in the reproductive tract of the guinea pig..Biol. Reprod. 1988; 38: 713-722Crossref PubMed Scopus (16) Google Scholar,92Coronel C.E. San Agustin J. Lardy H.A. Purification and structure of caltrin-like proteins from seminal vesicle of the guinea pig..J. Biol. Chem. 1990; 265: 6854-6859Abstract Full Text PDF PubMed Google Scholar). These separate sites of binding and function account for the fact that each of the GP caltrins inhibits about 50% of the calcium uptake that occurs in the absence of these seminal vesicle proteins (48Coronel C.E. Lardy H.A. Functional properties of caltrin proteins from seminal vesicles of the guinea pig..Mol. Reprod. Dev. 1992; 33: 74-80Crossref PubMed Scopus (28) Google Scholar).Figure 4Binding of guinea pig caltrin II to guinea pig epididymal sperm. Conditions are the same as described in the legend to Fig. 3 except that sperm were treated with purified guinea pig caltrin II and the rabbit antiserum had been prepared with caltrin II. A, immunofluorescence photomicrograph. Arrowsdesignate caltrin binding to the sperm tails. B, corresponding phase-contrast photomicrograph. Reprinted with permission from Ref. 48Coronel C.E. Lardy H.A. Functional properties of caltrin proteins from seminal vesicles of the guinea pig..Mol. Reprod. Dev. 1992; 33: 74-80Crossref PubMed Scopus (28) Google Scholar.View Large Image Figure ViewerDownload H" @default.
• W2093025897 created "2016-06-24" @default.
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• W2093025897 date "2003-02-01" @default.
• W2093025897 modified "2023-09-27" @default.
• W2093025897 title "Happily at Work" @default.
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