Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UNIPROT:P56851 (epididymal)
11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study provides evidence for the occurrence of a biochemical function for the polyamines, spermidine and spermine, in bovine epididymal spermatozoa. Methods are described for the detection and isolation of a protein kinase from spermatozoa which catalyzed transfer of phosphate from [gamma-32P]ATP to a unique endogenous protein of Mr = 70,000 in a reaction that was polyamine-dependent. Enzymatic phosphorylation of the 70,000-dalton protein by the purified protein kinase was sharply activated, at times more than 80-fold, by the polyamines, spermidine and spermine. Spermidine and spermine, together in equimolar combination, synergistically activated the protein kinase when compared with all other possible combinations of putrescine, spermidine, or spermine. The polyamine-dependent protein kinase was resolved by phosphocellulose chromatography into a catalytic component of Mr = 19,000 and a complex comprised of the catalytic component associated with the natural phosphate-acceptor protein of Mr = 70,000. This protein kinase was not activated by cyclic nucleotides or calcium ion.
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PMID:A polyamine-dependent protein kinase from bovine epididymal spermatozoa. 726 52

In the accompanying report (Visconti, P.E., Bailey, J.L., Moore, G.D., Pan, D., Olds-Clarke, P. and Kopf, G.S. (1995) Development, 121, 1129-1137) we demonstrated that the tyrosine phosphorylation of a subset of mouse sperm proteins of M(r) 40,000-120,000 was correlated with the capacitation state of the sperm. The mechanism by which protein tyrosine phosphorylation is regulated in sperm during this process is the subject of this report. Cauda epididymal sperm, when incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin do not display the capacitation-associated increases in protein tyrosine phosphorylation of this subset of proteins. This NaHCO3, CaCl2 or bovine serum albumin requirement for protein tyrosine phosphorylation can be completely overcome by the addition of biologically active, but not inactive, cAMP analogues. Addition of the active cAMP analogues to sperm incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin overcomes the inability of these media to support capacitation, as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. The effects of the cAMP analogues to enhance protein tyrosine phosphorylation and to promote capacitation appears to be at the level of the cAMP-dependent protein kinase (PKA), since two specific inhibitors of this enzyme (H-89 and Rp-cAMPS) block the capacitation-dependent increases in protein tyrosine phosphorylation in sperm incubated in media supporting capacitation. Capacitation, as assessed by the aforementioned endpoints, also appears to be inhibited by H-89 in a concentration-dependent manner. These results provide further evidence for the interrelationship between protein tyrosine phosphorylation and the appearance of the capacitated state in mouse sperm. They also demonstrate that both protein tyrosine phosphorylation and capacitation appear to be regulated by cAMP/PKA. Up-regulation of protein tyrosine phosphorylation by cAMP/PKA in sperm is, to our knowledge, the first demonstration of such an interrelationship between tyrosine kinase/phosphatase and PKA signaling pathways.
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PMID:Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. 753 69

A protein kinase that causes phosphorylation of serine and threonine residues of casein has been partially purified from goat cauda-epididymal sperm plasma membrane and characterized. The kinase, solubilized from the membrane with 1.0% Triton X-100, was purified to 480-fold by using DEAE-cellulose and casein-Sepharose affinity chromatographic techniques. The kinase is a strongly basic protein with pI of 9.5. The enzyme has a molecular mass of 310 kilodaltons as estimated by Sephacryl S-300 gel exclusion. The kinase showed affinity for protein substrates in the order membrane proteins > casein > phosvitin > histone > protamine. The apparent Km values of the kinase for casein and membrane proteins were 1 and 0.15 mg/mL, respectively. The synthetic peptides Kemptide and poly(Glu80Tyr20) did not serve as substrates of the enzyme. ATP, rather than GTP or PP(i), is the donor of phosphate for the phosphorylation reaction. Cyclic AMP and GMP, NaCl (0.25 M), KCl (0.25 M), Ca2+, calmodulin, phosphatidylserine, and muscle protein kinase inhibitor had no appreciable effect on the kinase activity. Heparin (0.5 microgram/mL) showed high affinity for inhibiting only 40% of the kinase activity, whereas polyamines at a relatively high concentration (5 mM) inhibited 40-50% of the enzymic activity. The kinase appears to be distinct from other protein kinases including casein kinases. The activity of the kinase derived from the purified sperm plasma membrane was markedly (approximately 90%) lost when the intact spermatozoa were pretreated with diazonium salt of sulfanilic acid, a membrane nonpenetrating surface probe.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of a protein kinase from goat sperm plasma membrane. 784 Sep 41

Based upon recent reports that the mRNA from the regulatory (R) RI beta subunit of cAMP-dependent protein kinase (PKA) was expressed in testicular extracts, we determined whether testicular extracts exhibited RI beta protein. To accomplish this goal, we initially determined the fundamental labeling and ionic characteristics of recombinant RI beta. Recombinant RI beta eluted from DEAE-cellulose with a salt concentration (of 0.075 M) equivalent to its elution position from soluble mouse brain extracts with catalytic subunit-free RI alpha. As predicted by its amino acid sequence homology to RI alpha, recombinant RI beta was not phosphorylated by PKA but was labeled specifically with 8-azido-adenosine 3':5'-[32P]monophosphate (8-N3[32P]cAMP). Additionally, RI antisera reacted equally with RI alpha (47 kDa) and recombinant RI beta (53 kDa). However, recombinant RI beta exhibited an unexpectedly basic pI of 6.65-6.85. By using a pH gradient for isoelectric focussing that allowed for clear focussing of 8-N3[32P]cAMP-labeled recombinant RI beta, 8-N3[32P]cAMP-labeled RI beta was readily detected by two-dimensional gel electrophoresis in rat brain particulate extracts and exhibited a pI equivalent to that of recombinant RI beta. The 53-kDa RI beta was undetectable either by its immunoreactivity or upon photoaffinity labeling with 8-N3[32P]cAMP by one or two-dimensional gel electrophoresis in soluble or particulate extracts of testes of 14-day-old, 45-day-old, or adult rats or in epididymal sperm. However, 8-N3[32P]cAMP-labeled RI beta was detected, albeit in very small levels, by two-dimensional electrophoresis upon separation of PKAs in testes of 14-day-old rats by DEAE-cellulose chromatography but was absent in equivalent extracts from adult rat testes. These results demonstrate that the unexpectedly basic pI of RI beta allows for its clear separation by two-dimensional electrophoresis from the RII proteins and therefore allows for its unambiguous identification. Further studies, however, are required to resolve the basis for the apparent disparity in testis RI beta mRNA and protein.
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PMID:Characterization of recombinant RI beta and evaluation of the presence of RI beta protein in rat brain and testicular extracts. 803 21

Cyclic AMP-dependent changes in phosphorylation of epididymal mouse sperm suspensions were examined in media designed to manipulate capacitation and the expression of parameters associated with full fertilizing ability, i.e. hyperactivated motility and the acrosome reaction. After initial assessment of cAMP-dependent protein kinase activity in frozen-thawed and lyophilized sperm suspensions using exogenous substrate, phosphorylation of endogenous sperm phosphoproteins was examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by autoradiography or immunoblotting. Numerous phosphoproteins were detected in both incapacitated and capacitated suspensions, the majority of which were probably concerned with motility; full expression of fertilizing ability appeared to involve an increase in the amount of endogenous phosphorylation as deduced from the decreased amount of 32P incorporation in these suspensions. The addition of the cAMP-dependent protein kinase inhibitors, H8 and PKI (6-22) amide, demonstrated that most of the phosphoproteins detected were phosphorylated in a cAMP-dependent manner. Of particular interest was a phosphoprotein with an M(r) of about 95,000 which was consistently observed in capacitated suspensions. Evidence suggests that this may be phosphorylated on tyrosine residues, since the inclusion of orthovanadate, a phosphoryltyrosine phosphatase inhibitor, altered phosphorylation of this protein. Furthermore, immunodetection using the antiphosphotyrosine antibody, PY-20, identified five proteins with approximate M(r) 116,000, 105,000, 95,000, 86,000, and 76,000, and possibly a sixth at 54,000. The 95,000 protein was consistently diminished in ionophore-treated spermatozoa, indicating that the protein was located in the acrosomal cap region. These results suggest that the protein may be the same phosphotyrosine-containing protein as that described by Leyton and Saling (1989) which has been proposed to play a role in acrosomal exocytosis.
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PMID:Cyclic AMP-dependent phosphorylation of epididymal mouse sperm proteins during capacitation in vitro: identification of an M(r) 95,000 phosphotyrosine-containing protein. 838 23

Activation of Ca2+ and cAMP-dependent Cl- conductances by extracellular ATP was studied using the whole-cell patch clamp technique. Immediately after addition of extracellular ATP (10 microM), activation of whole-cell Cl- current exhibiting delayed inactivation and activation kinetics at hyperpolarizing and depolarizing voltages, respectively, was observed. After prolonged activation, the kinetic characteristics of the ATP-induced Cl- current became time- and voltage-independent. When applied to the later phase of the ATP-activated whole-cell current, the disulfonic acid stilbene DIDS (200 microM) could only inhibit 64% of the current while diphenylamine-dicarboxylic acid (DPC, 1 mM) completely inhibited it. Inclusion of a peptide inhibitor for protein kinase A (PKI, 10 nM) in the pipette solution blocked ATP-induced time- and voltage-independent current activation but did not affect the delayed activating and inactivating current activation but did not affect the delayed activating and inactivating current which could be totally blocked by DIDS. Anion selectivity sequence was determined in the presence of either PKI or DIDS and found to be significantly different. Increased pipette EGTA (10 mM) or treatment of the cells with trifluoperazine (40 microM), an inhibitor of calmodulin, suppressed both types of ATP-induced Cl- currents. No current activation by ATP was observed when cells were dialyzed with the IP3 receptor blocker, heparin (10 ng/ml). These results suggest that extracellular ATP activates IP3-linked Ca(2+)-dependent regulatory pathway, which in turn activates cAMP-dependent pathway, leading to activation of both Ca2+ and cAMP-dependent Cl- conductances in epididymal cells.
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PMID:Extracellular ATP activates both Ca(2+)- and cAMP-dependent Cl- conductances in rat epididymal cells. 856 54

There is mounting evidence that in fat and other insulin-sensitive cells activation of protein synthesis may involve the dissociation of a protein (4E-BP1) from eukaryotic initiation factor (eIF)-4E thus allowing formation of the eIF-4F complex. This study compares the effects of insulin and epidermal growth factor (EGF) on the phosphorylation of 4E-BP1 in fat-cells (followed by gel-shift assays and incorporation of 32P) and on its association with eIF-4E. Several lines of evidence suggest that mitogenactivated protein kinase (MAP kinase) is not involved in these effects of insulin. Insulin causes much more extensive phosphorylation and dissociation of 4E-BP1 from eIF-4E than EGF, although EGF activates MAP kinase to a much greater extent than insulin. Moreover, MAP kinase does not phosphorylate 4E-BP1 when it is complexed with eIF-4E. In contrast, insulin activates the 40S ribosomal protein S6 kinase (p70S6K) 18-fold compared with a 2-fold activation by EGF, and the time course of this activation is similar to the phosphorylation and dissociation of 4E-BP1. Rapamycin, a specific inhibitor of the activation of this latter kinase, inhibits dissociation of 4E-BP1 from eIF-4E in cells incubated with insulin but reveals a phosphorylated from of 4E-BP1 which remains bound to eIF-4E. It is concluded that in rat epididymal fat-cells, the effects of insulin on 4E-BP1 involves multiple phosphorylation events. One phosphorylation event is rapamycin-insensitive, occurs only on bound 4E-BP1 and does not initiate dissociation. The second event does result in dissociation and is blocked by rapamycin, suggesting that the p70S6K signalling pathway is involved: p70S6K itself is probably not involved directly as this kinase does not phosphorylate 4E-BP1 in vitro.
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PMID:Both rapamycin-sensitive and -insensitive pathways are involved in the phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in response to insulin in rat epididymal fat-cells. 868 86

Acipimox is commonly used to treat hypertriglyceridaemia in non-insulin-dependent diabetic patients, but its precise mechanism of action has yet to be elucidated. We examined the in vitro effects of acipimox on the lipolytic regulatory cascade in epididymal adipocytes isolated from Wistar rats. Acipimox inhibited the lipolytic rate stimulated by adenosine deaminase (1 U/ml) in a concentration-dependent manner, reaching a near-basal value at 10 mumol/l acipimox. Lipolysis activated by sub-maximal levels of isoproterenol in combination with adenosine deaminase (20 mU/ml) was significantly (p < 0.05) decreased by 100 mumol/l acipimox, whereas, in the absence of adenosine deaminase, 100 mumol/l acipimox showed no significant (p > 0.05) inhibition. These findings suggested that the anti-lipolytic mechanism regulated by adenosine may also be regulated by acipimox. Acipimox diminished the intracellular cyclic AMP level produced by 25 nmol/l isoproterenol in the presence of adenosine deaminase (20 mU/ml) in a concentration-dependent manner. At the same level of stimulation, acipimox inhibited the cyclic AMP-dependent protein kinase activity ratio and lipolytic rate over the same concentration range, with significant (p < 0.05) reductions occurring at and above, 0.5 mumol/l and 10 mumol/l acipimox, respectively. Western blotting showed that upon lipolytic stimulation (1 U/ml adenosine deaminase; 100 nmol/l isoproterenol) a threefold increase in the lipolytic rate was accompanied by a significant (p < 0.05) rise in hormone-sensitive lipase associated with the lipid fraction. Acipimox (1 mmol/l) and insulin (1 nmol/l) re-distributed hormone-sensitive lipase back to the cytosol, with a corresponding significant (p < 0.05) loss from the fat cake fraction of adipocyte homogenates. In conclusion, the anti-lipolytic action of acipimox is mediated through suppression of intracellular cyclic AMP levels, with the subsequent decrease in cyclic AMP-dependent protein kinase activity, leading to the reduced association of hormone-sensitive lipase with triacylglycerol substrate in the lipid droplet of adipocytes.
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PMID:Mechanism of anti-lipolytic action of acipimox in isolated rat adipocytes. 872 Jun 2

Immotile bovine caput epididymal sperm contain levels of protein phosphatase activity twofold higher than do mature motile caudal sperm. Comparison of the inhibition profiles of endogenous phosphatase activities detected by okadaic acid (OA) and calyculin A (CA) revealed a pattern consistent with the predominance of a type 1 protein phosphatase (PP1). Immunoblot analysis identified PP1 gamma 2 (the testis-specific isoform of PP1) as the only PP1 isoform in sperm and showed little protein phosphatase 2A (PP2A). In addition, of the known PP1 inhibitors, i.e., DARPP-32, inhibitor 1 (I1), and inhibitor 2 (I2), only I2-like activity was detected in sperm. Inhibition of PP1 by the heat-stable I2-like activity purified from sperm could be reversed with purified glycogen synthase kinase-3 (GSK-3). Furthermore, sperm extracts contain an inactive complex of PP1 and I2 (termed PP1I) that could also be activated by purified GSK-3. The presence of GSK-3 in sperm was demonstrated by activation of purified PP1I, and quantitation revealed that immotile caput sperm contained sixfold higher GSK-3 activity than motile caudal sperm. Immunoblot analysis confirmed the expression of GSK-3 in sperm and revealed the occurrence of both the alpha and beta isoforms. Our findings suggest that the higher PP1 activity measured in immotile sperm, presumably due to higher GSK-3 activity, is responsible for holding motility in check. This conclusion was supported by the observation that the phosphatase inhibitors OA and CA, at micromolar and nanomolar levels, respectively, were able to induce motility in completely immotile bovine caput epididymal sperm and to stimulate the kinetic activity of mature caudal sperm. The intrasperm levels of cAMP, pH, and calcium were unaltered by treatment with these inhibitors. The results suggest a biochemical basis for the development and regulation of sperm motility and a possible physiological role for the PP1/I2/GSK-3 system.
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PMID:Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity. 883 95

The morphological and biochemical changes that occur in the haploid male germ cell during spermiogenesis facilitate the natural delivery of the paternally imprinted chromosomes into oocytes. Despite the obvious morphological changes, little is known about the molecular events underlying spermiogenesis. We recently cloned a novel 205-kDa manchette microtubule-associated serine/threonine protein kinase (MAST205) from mouse testis. The objective of this study was to further delineate the role of MAST205 in mammalian spermiogenesis. While MAST205 RNA levels were similar in pachytene spermatocytes, round spermatids, and residual bodies, MAST205 protein could be detected only in round spermatids and residual bodies. Kinase activity associated with MAST205 immunoprecipitates was low in pachytene spermatocytes, high in round spermatids, and maximal in residual bodies, indicating that MAST205-associated kinase activity is modified during spermatid maturation. Furthermore, MAST205 protein and the associated kinase activity were not detected in epididymal spermatozoa, indicating that MAST205 protein is either excluded from, or degraded in, the latter cell type. Multiple heterologous protein species were seen in immunoprecipitates from 35S-labeled mouse seminiferous tubules using an affinity-purified MAST205 antiserum. Consistent with this observation, MAST205 eluted as part of a 1-2 x 10(6) dalton protein complex when extracts of mouse testis were fractionated by Superose 6 column chromatography. MAST205 mRNA was detected in human testis indicative of conservation in other mammalian species. Taken together, these results indicate that the MAST205 complex functions in spermatid maturation in mammals.
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PMID:Increased activity associated with the MAST205 protein kinase complex during mammalian spermiogenesis. 890 15


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