Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.X200003200 on December 18, 2002

J. Biol. Chem., Vol. 278, Issue 6, 3499-3509, February 7, 2003
This Article
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/6/3499    most recent
X200003200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lardy, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lardy, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

REFLECTIONS
 Happily at Work

Henry Lardy

From the Institute for Enzyme Research, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53726

    INTRODUCTION
TOP
INTRODUCTION
The Caltrin Story
Dehydroepiandrosterone---A New...
REFERENCES

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 (1), 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 (2).

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 (3). 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 (4); cellular respiration rates varied with the availability of inorganic P and phosphate acceptor (5, 6); propionate was metabolized by CO2 addition to ultimately yield succinate (7, 8). 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 (9, 10) and a dozen that acted as ionophores (11), 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 (12, 13); the respiratory response was dependent on the utilization of acetylcarnitine (14). Thyroid hormone and also dehydroepiandrosterone induced the synthesis of mitochondrial glycerol-3-phosphate dehydrogenase to as much as 20 times the normal concentration (15-17) and formed part of a thermogenic system (17, 18). 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 (19, 20); serine was found to be converted to glucose by an entirely different pathway, probably the reverse of its synthesis from hydroxypyruvate (21). 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 (22); feeding tryptophan inhibits gluconeogenesis because its metabolite, quinolinate, forms a complex with ferrous ion that blocks PEPCK (23, 24). 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 (25, 26). 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 (2), 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.

    The Caltrin Story
TOP
INTRODUCTION
The Caltrin Story
Dehydroepiandrosterone---A New...
REFERENCES

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 (27). 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" (28).

During that same period we discovered that, unlike most substrates that increased both respiration and motility (29, 30), "beta -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" (29). This explanation seems also to apply to the working heart (31) and may have therapeutic implications (32).

Nearly a century ago the eminent physiologist Jacques Loeb demonstrated that fertilization of sea urchin eggs does not occur in the absence of Ca2+ (33, 90). 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 (34) and vertebrates (35, 36).

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 (35, 37, 91) when Donner Babcock found that the rapid uptake of calcium by bovine epididymal sperm did not occur in sperm separated from ejaculates (38). 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 beta -hydroxybutyrate (37, 38, 91). 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 (39) and the amino acid sequence was determined (Fig. 1) (40). 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 (41). 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 (42). Errors in the sequencing of seminal plasmin (43) were later corrected (44), 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.


View larger version (52K):
[in this window]
[in a new window]
  		Fig. 1.   The amino acid sequences of caltrins from bull, rat, guinea pig, and mouse.

On storage, caltrin lost activity as a blocker of calcium transport and became an enhancer of calcium uptake (45). This transformation could be accomplished rapidly by binding the fresh inhibitory caltrin to a cation exchanger. The recovered caltrin increased 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 (46).

With the aid of anti-bovine caltrin antiserum, two caltrin proteins were detected and isolated from guinea pig seminal vesicle secretions (47, 92). 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 (47, 92); bovine caltrin does not (39). 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 (48).

The seminal vesicles of rats and mice contain caltrins that have been purified and sequenced (Fig. 1) (49). Their calculated molecular weights are 6217 and 8476, respectively. Rat caltrin is derived from a 54-kDa inactive precursor produced in the seminal vesicles (50); the biosynthesis of rat caltrin and its precursor is androgen-dependent (51). 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 (52). 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 Mr 9600-10,500 by gel permeation and gel electrophoresis, but from its amino acid content we found a Mr 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 (53). It has 76 amino acids, 71 of which are identical with our sequence for mouse caltrin (49). Luo et al. (53) 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 (54) 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 (45). 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 (45). 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).


View larger version (140K):
[in this window]
[in a new window]
  		Fig. 2.   Bull sperm bind caltrin to the tail and over the acrosome. a, epididymal sperm treated with 0.40 mg of caltrin/108 cells in 1 ml and washed. c, ejaculated sperm washed free of seminal fluid and not exposed to purified caltrin. Sperm were spread and dried on glass slides, treated in succession with rabbit monospecific caltrin antiserum and goat anti-rabbit IgG that had been labeled with fluorescein isothiocyanate. Slides were washed to remove excess protein and viewed with a Zeiss fluorescence microscope (a and c); b and d are corresponding phase contrast photomicrographs. Epididymal sperm not exposed to caltrin did not bind the fluorescent-labeled antibody.

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/108 sperm) 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 (48). 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 (47, 92). 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 (48).


View larger version (92K):
[in this window]
[in a new window]
  		Fig. 3.   Binding of guinea pig caltrin I to guinea pig epididymal sperm. Epididymal sperm was treated with 0.5 mg of caltrin I/108 cells in 1 ml for 1 h and then washed twice with phosphate-buffered saline. Further treatments are described in the legend to Fig. 1 except that the antiserum had been prepared from rabbits treated with caltrin I. A, immunofluorescence photomicrograph. Arrows designate caltrin binding to the acrosome. B, corresponding phase-contrast photomicrograph.


View larger version (111K):
[in this window]
[in a new window]
  		Fig. 4.   Binding 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. Arrows designate caltrin binding to the sperm tails. B, corresponding phase-contrast photomicrograph. Reprinted with permission from Ref. 48.

The "Eureka!" announcement (55) that "observations of differences in the effect of seminal plasma contamination on hyperactivation and capacitation made in the present study provide further evidence for the existence of functionally separated, distinct regions in the spermatozoon" in effect confirmed for the human sperm what had been demonstrated much earlier in guinea pig sperm (48).

Some years ago Tschesche et al. (56) reported the presence of proteolytic inhibitory proteins in mammalian seminal plasma, and their observation has been confirmed by several laboratory groups. Rat caltrin and guinea pig caltrin I are also trypsin inhibitors (52), and the former is identical with the inhibitors from the pancreas isolated by Uda et al. (57) and from liver by Kido et al. (58). There is extensive homology between rat caltrin and trypsin inhibitors isolated from pancreatic secretions of many different mammalian species. Guinea pig caltrin II, bovine, and mouse caltrins are not trypsin inhibitors (50). The gene for caltrin (seminal plasmin) has been characterized (59) and, remarkably, has been shown to be a member of the extensive neuropeptide Y gene family (60). Seminal plasmin/caltrin was the subject of an excellent review (61).

In summary, the role of caltrins in fertilization can be postulated from their known functions. On ejaculation, spermatozoa bind caltrins as well as other seminal vesicle products. Bound caltrins prevent calcium movement into the acrosome and thus prevent a premature acrosome reaction. The hydrolytic and proteolytic enzymes are retained until needed. By preventing calcium uptake by the tail, caltrins keep the sperm moving forward. After some time the sperm will have moved up the female reproductive tract and encountered the egg(s); the phosphatidylserine will have been dissociated from the caltrin protein in the case of bovine and the carbohydrate residues hydrolytically removed in the case of rodents. The enhancer forms of caltrin then stimulate calcium uptake at the acrosome where it activates membrane discomposition and at the tail where it induces whiplash movement of the sperm tail. Species that produce two caltrins have one that acts at the acrosome and controls hyaluronidase and acrosin release; the other binds to the sperm tail and regulates motility.

Investigation of these proteins in our laboratory was dropped for lack of laboratory space on my reaching emeritus status in 1988 but is being continued by Carlos Coronel in Argentina.

    Dehydroepiandrosterone---A New Chapter
TOP
INTRODUCTION
The Caltrin Story
Dehydroepiandrosterone---A New...
REFERENCES

The University of Wisconsin treats its retirees more hospitably than Columbia treated Professor Chargaff (62); I was allowed to retain a small laboratory, enough for chemistry but not for extensive metabolic research.

A problem that was inviting concerned possible active steroid hormones derived metabolically from dehydroepiandrosterone (DHEA). This steroid was known as an intermediate in the conversion of cholesterol to testosterone and estrogens. Administered in large amounts, it caused fat and weight loss in obese mice (63), rats, and dogs, decreased blood sugar in diabetic mice (64), decreased the incidence of spontaneous and carcinogen-induced tumors in mice (65), enhanced immune responses (66), and improved memory in old mice (67). We, like some others, assumed that DHEA was converted metabolically to more active steroids that exerted these beneficial effects. Beginning in the 1960s many investigators had studied the conversion of DHEA to other steroids by animals, humans, and tissue preparations, but only a few of the products were tested for any biological activity. We initiated a program of synthesizing derivatives of DHEA that were logical metabolites in the hope of finding one or more new hormones. Such a search requires an assay for biological activity, and our earlier research provided one. Administering extra thyroid hormone to rats induces the formation of mitochondrial glycerophosphate dehydrogenase (GPDH) to 20 times the normal level in liver (15, 16) and somewhat less in other tissues (16). After Tagliaferro et al. (68) reported that DHEA enhanced metabolism and thermogenesis, we found this steroid induced the formation of hepatic GPDH but not that of other tissues (17, 69). Cytosolic malic enzyme is also increased by these hormones (70), and the response of these two enzymes to administered steroids thus provides a semiquantitative assay of activity. The two enzymes comprise a thermogenic system regulated by calcium and other factors (17, 18, 71, 72).

We found that hydroxylation of DHEA at any position other than 7 abolished the ability to increase the thermogenic enzymes (73). 7alpha -Hydroxy-, 7-oxo-, and 7beta -hydroxy-DHEA were more active than DHEA. Because activity increased in that sequence we postulated that the same sequence was involved in converting DHEA to an active hormone. By incubating DHEA with liver homogenate fortified with ATP, NADPH, and malate and assaying the products at short time intervals, that sequence was indeed established (74) and is shown in Fig. 5. The detection and quantitative measurement of the many products formed from DHEA (Fig. 5, 1) by liver were possible because of the analytical prowess of Dr. Ashok Marwah (75-79). Several additional products remain to be identified including some glucuronides.


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 5.   Steroids produced from DHEA (1) in liver homogenate fortified with ATP, NADPH, and malate. The broken arrows are postulated enzyme-catalyzed reactions.

DHEA derivatives bearing oxo- or hydroxyl groups at position 7 do not serve as precursors of androgens or estrogens and therefore are potential therapeutic agents. 7-Oxo-DHEA had no detectable toxicity in rats (80) or monkeys (81) even in massive doses and in a phase I clinical trial was well tolerated by normal men given doses up to 200 mg/day for 28 days (82). Tested at that dose for 8 weeks in obese subjects (body mass index of 31.9 ± 6.2 kg/m2) who were restricted to 1800 calories/day, subjects receiving 7-oxo-DHEA lost significantly more body weight and fat than those receiving placebos (83). 7-Oxo-DHEA was far more effective than DHEA as an enhancer of memory in old mice and in restoring memory in mice treated with scopolamine (84). Androstenediol (Fig. 5, 6), one of the main products of DHEA metabolism in liver, has been known for many years to have estrogen activity. It also activates androgen receptor transcriptional activity in prostate cancer cells (85). This function is not inhibited by hydroxyflutamide or bicalutamide, two agents used for treating prostate cancer (86). This raises the question whether androstenediol, produced in adrenals and liver, accounts for the failure of orchidectomy to be an effective long term treatment for prostatic cancer. In a collaborative study, we have also found that DHEA has activity in adipose cells not displayed by its metabolites (87).

Structure/activity comparisons show that ring D of DHEA can be altered in several different ways without abolition of activity. The ring can be expanded by insertion of oxygen at 17a (88) with retention of the ability to induce the formation of both GPDH and malic enzyme. Hydroxylation at position 15 or introduction of 15-16 unsaturation, nearly abolishes the response of GPDH, but induction of malic enzyme is retained (89). Substitutions at position 16 yield steroids with varying activity; some are highly active and point the way to possible routes to true hormones. The search goes on.

    FOOTNOTES

Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.X200003200

Address correspondence to: halardy{at}facstaff.wisc.edu.

    REFERENCES
TOP
INTRODUCTION
The Caltrin Story
Dehydroepiandrosterone---A New...
REFERENCES

1. Kornberg, A. (2001) Remembering our teachers. J. Biol. Chem. 276, 3-11[Free Full Text]
2. Lardy, H. A. (1985) in A half century of biochemistry. In A History of Biochemistry (Semenza, G., ed) , pp. 297-325, Elsevier Science Publishing Co., Inc., New York
3. Lardy, H., Wiebelhaus, V., and Mann, K. (1950) The mechanism by which glyceraldehyde inhibits glycolysis. J. Biol. Chem. 187, 325-337[Free Full Text]
4. Lardy, H., Potter, R., and Elvehjem, C. (1947) The role of biotin in bicarbonate utilization by bacteria. J. Biol. Chem. 169, 451-452[Free Full Text]
5. Lardy, H. (1952) The role of phosphate in metabolic control mechanisms. The Biology of Phosphorus , pp. 287-294, Michigan State College Press, East Lansing, MI
6. Lardy, H., and Wellman, H. (1952) Oxidative phosphorylations: role of inorganic phosphate and acceptor systems in control of metabolic rates. J. Biol. Chem. 195, 215-224[Free Full Text]
7. Lardy, H., and Peanasky, R. (1953) Metabolic effects of biotin. Physiol. Rev. 33, 560-565[Free Full Text]
8. Lardy, H., and Adler, J. (1956) Synthesis of succinate from propionate and bicarbonate by soluble enzymes from liver mitochondria. J. Biol. Chem. 219, 933-942[Free Full Text]
9. Lardy, H., Reed, P., and Lin, C.-H. (1975) Antibiotic inhibitors of mitochondrial ATP synthesis. Fed. Proc. 34, 1707-1710[Medline] [Order article via Infotrieve]
10. Lardy, H. (1980) Antibiotic inhibitors of mitochondrial energy transfer. Pharmacol. Ther. 11, 649-660[CrossRef][Medline] [Order article via Infotrieve]
11. Lardy, H., Graven, S., and Estrada-O, S. (1967) Specific induction and inhibition of cation and anion transport in mitochondria. Fed. Proc. 26, 1355-1360[Medline] [Order article via Infotrieve]
12. Garbers, D., Lust, W., First, N., and Lardy, H. (1971) Effects of phosphodiesterase inhibitors and cyclic nucleotides on sperm respiration and motility. Biochemistry 10, 1825-1831[CrossRef]
13. Garbers, D., First, N., and Lardy, H. (1973) The stimulation of bovine sperm metabolism by cyclic nucleotide phosphodiesterase inhibitors. Biol. Reprod. 8, 589-598[Abstract]
14. Milkowski, A., Babcock, D., and Lardy, H. (1976) Activation of bovine epididymal sperm respiration by caffeine. Arch. Biochem. Biophys. 176, 250-256[CrossRef][Medline] [Order article via Infotrieve]
15. Lee, Y.-P., Takemori, A., and Lardy, H. (1959) Enhanced oxidation of alpha -glycerophosphate by mitochondria of thyroid-fed rats. J. Biol. Chem. 234, 3051-3054[Free Full Text]
16. Lee, Y.-P., and Lardy, H. (1965) Influence of thyroid hormones on L-alpha -glycerophosphate dehydrogenasees and other dehydrogenases in various organs of the rat. J. Biol. Chem. 240, 1427-1436[Free Full Text]
17. Lardy, H., Su, C.-Y., Kneer, N., and Wielgus, S. (1989) in Dehydroepiandrosterone induces enzymes that permit thermogenesis and decrease metabolic efficiency. In Hormones, Thermogenesis, and Obesity (Lardy, H. , and Stratman, F., eds) , pp. 415-426, Elsevier Science Publishers B.V., Amsterdam
18. Lardy, H. (1999) in Dehydroepiandrosterone and ergosteroids affect energy expenditure. In Health Promotion and Aging (Watson, R. R., ed) , pp. 33-42, Harwood, Amsterdam
19. Lardy, H., Paetkau, V., and Walter, P. (1965) Paths of carbon in gluconeogenesis and lipogenesis: I. The role of mitochondria in supplying precursors of phosphoenolpyruvate. Proc. Natl. Acad. Sci. U. S. A. 53, 1410-1415[Free Full Text]
20. Walter, P., Paetkau, V., and Lardy, H. (1966) Paths of carbon in gluconeogenesis and lipogenesis: III. J. Biol. Chem. 241, 2523-2532[Abstract/Free Full Text]
21. Lardy, H., Veneziale, C., and Gabrielli, F. (1969) Paths of carbon in gluconeogenesis. FEBS Symp. 19, 55-62
22. Merryfield, M., and Lardy, H. (1982) Ca2+-mediated activation of phosphoenolpyruvate carboxykinase occurs via the release of Fe2+ from rat liver mitochondria. J. Biol. Chem. 257, 3628-3635[Abstract/Free Full Text]
23. Veneziale, C., Walter, P., Kneer, N., and Lardy, H. (1967) Influence of tryptophan and its metabolites on gluconeogenesis in the isolated, perfused liver. Biochemistry 6, 2129-2138[CrossRef][Medline] [Order article via Infotrieve]
24. Snoke, R., Johnston, J., and Lardy, H. (1971) Response of phosphopyruvate carboxylase to tryptophan metabolites and metal ions. Eur. J. Biochem. 24, 342-346[Medline] [Order article via Infotrieve]
25. Bobyleva, V., and Lardy, H. (1986) The role of malate in exercise-induced enhancement of mitochondrial respiration. Arch. Biochem. Biophys. 245, 470-476[CrossRef][Medline] [Order article via Infotrieve]
26. Bobyleva, V., Wehbie, R., and Lardy, H. (1986) The role of malate in hormone-induced enhancement of mitochondrial respiration. Arch. Biochem. Biophys. 245, 477-482[CrossRef][Medline] [Order article via Infotrieve]
27. Lardy, H. A., and Phillips, P. H. (1939) Preservation of spermatozoa. Proc. Am. Soc. Animal Production 32, 219-221
28. Lardy, H. A., and Phillips, P. H. (1943) The effect of thyroxine and dinitrophenol on sperm metabolism. J. Biol. Chem. 149, 177-182[Free Full Text]
29. Lardy, H. A., Hansen, R. G., and Phillips, P. H. (1945) The metabolism of bovine epididymal spermatozoa. Arch. Biochem. 6, 41-51
30. Lardy, H. A., and Phillips, P. H. (1945) Studies of fat and carbohydrate oxidation in mammalian spermatozoa. Arch. Biochem. 6, 53-61
31. Sato, K., Kashiwaya, Y., Keon, C. A., Tsuchiya, N., King, M. T., Radda, G. K., Chance, B., Clarke, K., and Veech, R. L. (1995) Insulin, ketone bodies and mitochondrial energy transduction. FASEB J. 9, 651-658[Abstract]
32. Veech, R., Chance, B., Kashiwaya, Y., Lardy, H. A., and Cahill, G. F. (2001) Ketone bodies, potential therapeutic uses. IUBMB Life 51, 241-247[Medline] [Order article via Infotrieve]
33. Loeb, J. (1915) On the nature of the conditions which determine or prevent the entrance of the spermatozoan into the egg. Am. Natur. 49, 257-285[CrossRef]
34. Dan, J. C. (1954) Studies on the acrosome. III. Effect of calcium deficiency. Biol. Bull. 107, 335-349[Abstract/Free Full Text]
35. Babcock, D. F., First, N. L., and Lardy, H. A. (1976) Action of the ionophore A 23187 at the cellular level. J. Biol. Chem. 251, 3881-3886[Abstract/Free Full Text]
36. Yanagimachi, R. (1978) Calcium requirement for sperm-egg fusion in mammals. Biol. Reprod. 19, 949-958[Abstract]
37. Singh, J. P., Babcock, D. F., and Lardy, H. A. (1978) Increased calcium-ion influx is a component of capacitation of spermatozoa. Biochem. J. 172, 549-556[Medline] [Order article via Infotrieve]
38. Babcock, D. E., Singh, J. P., and Lardy, H. A. (1979) Alteration of membrane permeability to calcium ions during maturation of bovine spermatozoa. Dev. Biol. 69, 85-93[CrossRef][Medline] [Order article via Infotrieve]
39. Rufo, G. A., Singh, J. B., Babcock, D. E., and Lardy, H. A. (1982) Purification and characterization of a calcium transport inhibitor protein from bovine seminal plasma. J. Biol. Chem. 257, 4627-4632[Abstract/Free Full Text]
40. Lewis, R. V., San Agustin, J., Kruggel, W., and Lardy, H. A. (1985) The structure of caltrin, the calcium-transport inhibitor of bovine seminal plasma. Proc. Natl. Acad. Sci. U. S. A. 82, 6490-6491[Abstract/Free Full Text]
41. Rufo, G. A., Schoff, P. K., and Lardy, H. A. (1984) Regulation of calcium content in bovine spermatozoa. J. Biol. Chem. 259, 2547-2552[Abstract/Free Full Text]
42. Reddy, E. S. P., and Bhargava, P. M. (1979) Seminal plasmin---an antimicrobial protein from bovine seminal plasma which acts in E. coli by specific inhibition of rRNA synthesis. Nature 279, 725-728[CrossRef][Medline] [Order article via Infotrieve]
43. Theil, R., and Scheit, K. H. (1983) Amino acid sequence of seminal plasmin, an antimicrobial protein from bull semen. EMBO J. 12, 1159-1163
44. Sitaram, N., Kumari, V. K., and Bhargava, P. M. (1986) Seminal plasmin and caltrin are the same protein. FEBS Lett. 201, 233-236[CrossRef][Medline] [Order article via Infotrieve]
45. San Agustin, J., Hughes, P., and Lardy, H. A. (1987) Properties and function of caltrin, the calcium-transport inhibitor of bull seminal plasma. FASEB J. 1, 60-66[Abstract]
46. San Agustin, J., and Lardy, H. A. (1990) Bovine seminal plasma constituents modulate the activity of caltrin, the calcium-transport regulating protein of bovine spermatozoa. J. Biol. Chem. 265, 6860-6867
47. Coronel, C. E., San Agustin, J., and Lardy, H. A. (1988) Identification and partial characterization of caltrin-like proteins in the reproductive tract of the guinea pig. Biol. Reprod. 38, 713-722[Abstract]
48. Coronel, C. E., and Lardy, H. A. (1992) Functional properties of caltrin proteins from seminal vesicles of the guinea pig. Mol. Reprod. Dev. 33, 74-80[CrossRef][Medline] [Order article via Infotrieve]
49. Coronel, C. E., Winnica, D. E., Novella, M. L., and Lardy, H. A. (1992) Purification, structure, and characterization of caltrin proteins from seminal vesicle of the rat and mouse. J. Biol. Chem. 267, 20909-20915[Abstract/Free Full Text]
50. Coronel, C. E., Novella, M. L., Winnica, D. E., and Lardy, H. (1993) Isolation and characterization of a 54-kilodalton precursor of caltrin, the calcium transport inhibitor protein from seminal vesicles of the rat. Biol. Reprod. 48, 1326-1333[Abstract]
51. Novella, M. L., Maldonado, C., Aoki, A., and Coronel, C. E. (1999) Androgen-dependent synthesis/secretion of caltrin, calcium transport inhibitor protein of mammalian seminal vesicle. Arch. Androl. 42, 1-12[CrossRef][Medline] [Order article via Infotrieve]
52. Winnica, D. E., Novella, M. L., Dematteis, A., and Coronel, C. E. (2000) Trypsin/acrosin inhibitor activity of rat and guinea pig caltrin proteins. Structural and functional studies. Biol. Reprod. 63, 42-48[Abstract/Free Full Text]
53. Luo, C-W., Lin, H-J., and Chen, Y-H. (2001) A novel heat-labile phospholipid-binding protein, SVS VII, in mouse seminal vesicle as a sperm motility enhancer. J. Biol. Chem. 276, 6913-6921[Abstract/Free Full Text]
54. Chen, L.-Y., Lin, Y.-H., Lai, M.-L., and Chen, Y.-H. (1998) Developmental profile of a caltrin-like protease inhibitor, P12, in mouse seminal vesicle and characterization of its binding sites on sperm surface. Biol. Reprod. 59, 1498-1505[Abstract/Free Full Text]
55. Mortimore, S. T., Swan, M. A., and Mortimore, D. (1998) Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa. Hum. Reprod. 13, 2139-2146[Abstract/Free Full Text]
56. Tschesche, H., Kupfer, S., Lengel, O., Klauser, R., Meier, M., and Fritz, H. (1974) Proteinase inhibitors. Second International Research Conference. Bayer Symposium V , pp. 164-187, Springer-Verlag, Berlin
57. Uda, K., Ogawa, M., Shibata, T., Murata, A., Mori, T., Kikuchi, N., Toshida, N., Tsunasawa, S., and Sakiyama, F. (1988) Purification, characterization and amino-acid sequencing of two pancreatic secretory trypsin inhibitors in rat pancreatic juice. Biol. Chem. Hoppe-Seyler 369 (suppl.), 55-61
58. Kido, H., Yokogoshi, Y., and Katunuma, N. (1990) A low-molecular-mass Kazal-type protease inhibitor isolated from rat hepatocytes is identical to rat pancreatic secretory trypsin inhibitor. II. Eur. J. Biochem. 188, 501-506[Medline] [Order article via Infotrieve]
59. Kuhlmann, J. K., and Scheit, K. H. (1993) Characterization of the gene for seminal plasmin, a secretory protein of the bovine seminal vesicle. Biochim. Biophys. Acta 1173, 85-86[Medline] [Order article via Infotrieve]
60. Herzog, H., Hort, Y., Schneider, R., and Shine, J. (1995) Seminal plasmin: recent evolution of another member of the neuropeptide Y gene family. Proc. Natl. Acad. Sci. U. S. A. 92, 594-598[Abstract/Free Full Text]
61. Sitaram, N., and Nagara, R. (1995) Seminal plasmin. Bioessays 17, 415-421[CrossRef][Medline] [Order article via Infotrieve]
62. Chargaff, E. (1978) Heraclitean Fire , Rockefeller University Press, New York
63. Yen, T. T., Allen, J. A., Pearson, D. V., Acton, J., and Greenberg, M. M. (1977) Lipids 12, 409-413[Medline] [Order article via Infotrieve]
64. Coleman, D. L., Schwizer, R., and Leiter, E. (1984) Effect of genetic background on the therapeutic effects of dehydroepiandrosterone (DHEA) in diabetes-obesity mutants and in aged normal mice. Diabetes 33, 26-32[Abstract]
65. Schwartz, A. G., and Tannen, R. H. (1981) Inhibition of 7,12-dimethylbenzanthracene and urethane-induced lung tumor formation in A/J mice by long-term treatment with dehydroepiandrosterone. Carcinogenesis 2, 1335-1337[Abstract/Free Full Text]
66. Loria, R., Inge, T., Cook, S. S., Szakal, A., and Regelson, W. (1988) Protection against acute lethal viral infections with the native steroid dehydroepiandrosterone(DHEA). Med. Virol. 26, 301-314
67. Flood, J. F., Morley, J. E., and Roberts, E. (1992) Memory enhancing effects in male mice of pregnenolone and steroids metabolically derived from it. Proc. Natl. Acad. Sci. U. S. A. 89, 1567-1571[Abstract/Free Full Text]
68. Tagliaferro, A., Davis, J. R., Truchon, S., and Van Hamont, N. (1986) Effects of dehydroepiandrosterone acetate on metabolism, body weight and composition of male and female rats. J. Nutr. 116, 1977-1983[Abstract/Free Full Text]
69. Su, C.-Y., and Lardy, H. (1991) Induction of hepatic mitochondrial glycerophosphate dehydrogenase in rats by dehydroepiandrosterone. J. Biochem. (Tokyo) 110, 207-213[Abstract/Free Full Text]
70. Tepperman, H., de la Garza, S., and Tepperman, J. (1968) Effect of dehydroepiandrosterone and diet protein on liver enzymes and lipogenesis. Am. J. Physiol. 214, 1126-1132[Free Full Text]
71. Wernette, M., Ochs, R. S., and Lardy, H. (1981) Ca2+ stimulation of rat liver mitochondrial glycerophosphate dehydrogenase. J. Biol. Chem. 256, 12767-12771[Free Full Text]
72. Lardy, H., Kneer, N., Bellei, M., and Bobyleva, V. (1995) Induction of thermogenic enzymes by DHEA and its metabolites. Ann. N. Y. Acad. Sci. 774, 171-179[CrossRef][Medline] [Order article via Infotrieve]
73. Lardy, H., Kneer, N., Wei, Y., Partridge, B., and Marwah, P. (1998) Ergosteroids II: biologically active metabolites and synthetic derivatives of dehydroepiandrosterone. Steroids 63, 158-165[CrossRef][Medline] [Order article via Infotrieve]
74. Marwah, A., Marwah, P., and Lardy, H. (2002) Ergosteroids VI: metabolism of dehydroepiandrosterone by rat liver in vitro: a liquid chromatographic-mass spectrometric study. J. Chromatogr. B Biomed. Appl. 767, 285-299[CrossRef]
75. Marwah, A., Marwah, P., and Lardy, H. (1999) Development and validation of a high-performance liquid chromatography assay for the quantitative determination of 7-oxo-dehydroepiandrosterone in human plasma. J. Chromatogr. B Biomed. Appl. 721, 197-205[CrossRef]
76. Marwah, A., Marwah, P., and Lardy, H. (2001) Liquid chromatography-electrospray ionization mass spectrometric analysis of corticosterone in rat plasma using selected ion monitoring. J. Chromatog. B Biomed. Appl. 757, 333-342[CrossRef]
77. Marwah, A., Marwah, P., and Lardy, H. (2001) High performance liquid chromatographic analysis of dehydroepiandrosterone. J. Chromatogr. A 935, 279-296[CrossRef][Medline] [Order article via Infotrieve]
78. Marwah, A., Marwah, P., and Lardy, H. (2002) Ergosteroids VII: perchloric acid induced transformations of 7-oxygenated steroids and their bio-analytical applications; a liquid chromatographic-mass spectrometry study. J. Bioorganic Chem. 30, 233-248
79. Marwah, A., Marwah, P., and Lardy, H. (2002) Ergosteroids VIII. Enhancement of signal response of neutral steroidal compounds in liquid chromatographic-electrospray ionization mass spectrometric analysis by mobile phase additives. J. Chromatogr. A. 964, 137-151[CrossRef][Medline] [Order article via Infotrieve]
80. Lardy, H., Henwood, S., and Weeks, C. (1999) An acute oral gavage study of 3beta -acetoxyandrost-5-ene-7,17-dione in rats. Biochem. Biophys. Res. Commun. 254, 120-123[CrossRef][Medline] [Order article via Infotrieve]
81. Henwood, S., Weeks, C., and Lardy, H. (1999) An escalating dose oral gavage study of 3beta -acetoxyandrost-5-ene-7,17-dione in Rhesus monkeys. Biochem. Biophys. Res. Commun. 254, 124-126[CrossRef][Medline] [Order article via Infotrieve]
82. Davidson, M., Marwah, A., Sawchuk, R., Maki, K., Marwah, P., Weeks, C., and Lardy, H. (2002) Safety and pharmacokinetic study with escalating doses of 3-acetyl-7-oxo-dehydroepiandrosterone in healthy male volunteers. Clin. Invest. Med. 23, 300-310
83. Kalman, D., Colker, C., Swain, M., Torina, G., and Shi, Q. (2002) A randomized double-blind, placebo-controlled study of 3-acetyl-7-oxo-dehydroepiandrosterone in healthy overweight adults. Curr. Ther. Res. 61, 435-442[CrossRef]
84. Shi, J., Schulze, S., and Lardy, H. (2000) The effect of 7-oxoDHEA acetate on memory in young and old 57BL/6 mice. Steroids 63, 124-129
85. Miyamoto, H., Yeh, S., Lardy, H., Messing, E., and Chang, C. (1998) Delta 5-Androstenediol is a natural hormone with androgenic activity in human prostate cancer cells. Proc. Natl. Acad. Sci. U. S. A. 95, 11083-11088[Abstract/Free Full Text]
86. Prostate Cancer Trialists' Collaborative Group. (1995) Lancet 346, 265-269[Medline] [Order article via Infotrieve]
87. Gomez, F. E., Miyazaki, M., Kim, Y.-C., Marwah, P., Lardy, H., Ntambi, J., and Fox, B. G. (2002) Molecular differences caused by differentiation of 3T3-L1 preadipocytes in the presence of either dehydroepiandrostrerone (DHEA) or 7-oxo-DHEA. Biochemistry 41, 5473-5482[CrossRef][Medline] [Order article via Infotrieve]
88. Reich, I., Lardy, H., Wei, Y., Marwah, P., Kneer, N., Powell, D. R., and Reich, H. J. (1998) Ergosteroids III. Synthesis and biological activity of seco-steroids related to dehydroepiandrosterone. Steroids 63, 542-553[CrossRef][Medline] [Order article via Infotrieve]
89. Reich, I., Reich, H., Kneer, N., and Lardy, H. (2002) Ergosteroids V: preparation and biological activity of various D-ring derivatives in the 7-oxo-dehydroepiandrosterone series. Steroids 67, 221-233[CrossRef][Medline] [Order article via Infotrieve]
90. Loeb, J. (1913) Artificial Parthenogenesis and Fertilization , University of Chicago Press, Chicago, IL
91. Singh, J. P., Babcock, D. F., and Lardy, H. A. (1980) Induction of accelerated acrosome reaction in guinea pig sperm. Biol. Reprod. 22, 566-570[Abstract]
92. Coronel, C. E., San Agustin, J., and Lardy, H. A. (1990) Purification and structure of caltrin-like proteins from seminal vesicle of the guinea pig. J. Biol. Chem. 265, 6854-6859[Abstract/Free Full Text]

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol Biol EvolHome page
R. C. Karn, N. L. Clark, E. D. Nguyen, and W. J. Swanson
Adaptive Evolution in Rodent Seminal Vesicle Secretion Proteins
Mol. Biol. Evol., November 1, 2008; 25(11): 2301 - 2310.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
A. Dematteis, S. D. Miranda, M. L. Novella, C. Maldonado, R. H. Ponce, J. A. Maldera, P. S. Cuasnicu, and C. E. Coronel
Rat Caltrin Protein Modulates the Acrosomal Exocytosis During Sperm Capacitation
Biol Reprod, September 1, 2008; 79(3): 493 - 500.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Kresge, R. D. Simoni, and R. L. Hill
Henry Lardy's Contributions to Understanding the Metabolic Pathway
J. Biol. Chem., May 20, 2005; 280(20): e17 - e17.
[Full Text] [PDF]

This Article
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/6/3499    most recent
X200003200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lardy, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lardy, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement