Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The involvement of superoxide anion (O2o-) in human sperm capacitation and/or acrosome reaction was investigated. Addition of superoxide dismutase (SOD) to the medium at the beginning of the capacitation process or 15 min before induction of the acrosome reaction, decreased the level of ionophore-induced acrosome reaction. Hyperactivation was unaffected by the presence of SOD during the capacitation process. Addition of calcium ionophore to the sperm suspension increased production of O2o- by the spermatozoa by four to five-fold and induced the acrosome reaction. In the presence of SOD, superoxide anion could not be detected in the medium and the rate of induced-acrosome reaction was decreased greatly. The presence of an inhibitor of protein kinase C inhibited the production of O2o- in the medium and reduced the induced-acrosome reaction. The production of O2o- and the acrosome reaction were also increased by exposure of spermatozoa to 12-myristate 13-acetate phorbol ester, a specific activator of protein kinase C. While the level of spontaneous acrosome reaction was not increased by the direct addition of O2o- to the medium, its presence induced the release of unesterified fatty acids from membrane phospholipids. These findings suggest that the production of O2o- by spermatozoa could be involved in the ionophore-induced acrosome reaction, possibly through the de-esterification of membrane phospholipids. However, this production of superoxide anion is not sufficient on its own to induce the acrosome reaction.
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PMID:Superoxide anion production by human spermatozoa as a part of the ionophore-induced acrosome reaction process. 766 12

The human acrosome reaction (AR; sperm exocytosis) is absolutely required for fertilization. In the course of further characterizing the AR and its control, an AR-inhibiting glycoprotein (ARIG) from human seminal plasma was purified by differential centrifugation, carboxymethyl cellulose chromatography, chromatofocusing, and Sephacryl S300 gel filtration. A highly purified protein with a molecular weight of 74,000 was obtained as determined by gel filtration and SDS-PAGE. ARIG eluted in a narrow pH range (6.2-5.4) during chromatofocusing, corresponding to a pl of 5.8 +/- 0.4. It had covalent modifications, including internal disulfide bonds, and both complex N-linked and O-linked oligosaccharide chains. Lectin analysis suggested that sialic acid was absent and that the complex oligosaccharide chains had sequences containing galactose, galactosamine, and/or glucosamine in a beta 1-4 linkage. Mannose residues were also present. When ARIG was added to in vitro-capacitated human spermatozoa 30 min prior to the calcium ionophore A23187, the AR was significantly inhibited (ID50 = 8.5 micrograms/ml). In addition, ARIG reduced sperm exocytosis in response to atrial natriuretic peptide (a guanylate cyclase activator) and to the protein kinase C activators phorbol myristate acetate and dioctanoylglycerol. The ability of ARIG to block the human AR induced by a variety of agonists and the fact that biological activity of the protein was lost after removal of its sugar moieties suggests that it may function as a general inhibitor of sperm exocytosis and that its interaction with spermatozoa may be mediated by carbohydrate-binding proteins on the sperm cell.
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PMID:Purification and partial characterization of acrosome reaction inhibiting glycoprotein from human seminal plasma. 766 49

The influence on spermine on the acrosomal exocytosis of capacitated bovine spermatozoa was studied. Dual effect of spermine was observed, depending on its concentration. It was shown that 10 microM spermine stimulated acrosomal exocytosis and prostaglandin F2 alpha production, whereas higher concentrations of spermine inhibited these processes. Acrosomal exocytosis induced by spermine was inhibited by staurosporine, a specific protein kinase C (PKC) inhibitor, indicating that PKC may be involved in this stimulation. Also, acrosomal exocytosis induced by the PKC activator phorbol 12-myristate-13-acetate was inhibited by 10 mM spermine. Therefore, these data indicate that spermine is involved in signal transduction events leading to exocytosis. We suggest that the concentration-dependent reversal of the stimulatory action of spermine could be explained by the existence of two binding sites for spermine: high affinity sites involved in inducing acrosomal exocytosis by low spermine concentration and low affinity sites mediating inhibition of acrosomal exocytosis by high concentration of spermine.
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PMID:Dual effect of spermine on acrosomal exocytosis in capacitated bovine spermatozoa. 774 86

The human sperm acrosome reaction (AR) occurs via the activation of at least two signal transduction pathways. The purpose of this investigation was to characterize two of the pathways, the protein kinase A (PKA) and C (PKC) pathways, and determine whether pathway "crosstalk" occurs between them in eliciting the AR in capacitated spermatozoa. Stimulators of each pathway were tested in a dose-dependent manner. ARmax, ED50, and delta ARmax (%ARmax-%ARcontrol) values were calculated. The PKA pathway stimulators forskolin and dibutyryl cyclic AMP (dbcAMP) induced an ARmax at 1.0 microM and 1.0 mM, respectively. The ED50 and delta ARmax values were: 0.01 microM and 17% for forskolin and 0.069 mM and 13% for dbcAMP. Two stimulator types of the PKC pathway were tested: synthetic diacylglycerols (DG) and a phorbol diester. 1,2-dioleoyl-sn-glycerol and 1,2-dioctanoyl-sn-glycerol, analogues of the PKC-activating second messenger DG, each induced an ARmax at 50 microM. The ED50 and delta AR max values were: 33 microM and 24% for 1,2-dioleoyl and 34.8 microM and 34% for 1,2-dioctanoyl. 4 beta-Phorbol-12,13-didecanoate, a PKC stimulator, induced an ARmax at 0.1 microM. The ED50 and delta ARmax were 0.021 microM and 26%. An inhibitor of each kinase was added at the end of the capacitation period and prior to stimulation by inducers at their ARmax dose. KT5720, a PKA inhibitor, caused a dose-dependent reduction of the forskolin and dbcAMP-induced AR. Calphostin C, a PKC inhibitor, prevented stimulation of the AR by 1,2-dioleoyl and 4 beta-phorbol-12,13-didecanoate. To investigate pathway "crosstalk," the following experiments were conducted: (1) stimulators of each pathway were combined and tested at the ARmax and ED50 concentrations for each; (2) spermatozoa were pretreated with a kinase inhibitor and then stimulated using an alternative pathway stimulator; and (3) a PKA or PKC inhibitor and a combination of PKA and PKC stimulators, at ED50 concentrations, were tested. The results for (1) indicate an additive AR response of ED50 concentrations but not for ARmax doses. The results for (2) demonstrate that a kinase inhibitor for one pathway prevents induction of the AR by a stimulator of the alternative pathway. Finally, the results for (3) show that a kinase inhibitor for one pathway prevents induction of the AR by the combined use of separate pathway stimulators. When taken collectively, the present results suggest a convergent mechanism of crosstalk between the PKA and PKC pathways leading to the human sperm AR.
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PMID:Characterization of two second messenger pathways and their interactions in eliciting the human sperm acrosome reaction. 776 51

Ejaculated bovine spermatozoa were examined for their capacity to synthesize prostaglandins E2 and F2 alpha (PGE2, PGF2 alpha). It was found that in the absence of exogenous substrate, arachidonic acid, basal PGF2 alpha production was less than that of PGE2. However, addition of 61 mumol arachidonic acid I-1 resulted in at least a twofold increase in PGE2 and PGF2 alpha above control values (1.3 ng and 0.3 ng per 10(8) spermatozoa, respectively). Addition of calcium and the calcium ionophore A23187 to the incubation medium did not cause a significant increase in the production of either PG. The presence of indomethacin (100-200 micrograms ml-1) caused a 50-70% inhibition of the production of both PGs. Activity of cyclooxygenase was determined by western blot analysis, using a specific polyclonal antiserum, and by fluorescence immunohistochemistry using a monoclonal antibody. The western blot displayed a clear signal for the presence of cyclooxygenase in ejaculated and epididymal spermatozoa. The immunohistochemical studies showed that the enzyme is localized in the apical region of the head, the post-acrosomal region and the mid-piece of the tail. Since the synthesis of PGs in the absence of exogenous arachidonic acid is low, the effect of melittin, a known phospholipase A2 activator, on PG production was examined. Incubation of spermatozoa with melittin produced a threefold increase in PGE2 and a sixfold increase in PGF2 alpha. Staurosporine, a protein kinase C inhibitor, inhibited the effect of melittin indicating that activation of phospholipase A2 by protein kinase C is an obligatory step in PG synthesis by bovine spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Localization of cyclooxygenase and production of prostaglandins in bovine spermatozoa. 793 76

The presence and possible role of protein kinase C in the regulation of fowl sperm functions were investigated. Immunoblot analysis of sperm extract using antibody to protein kinase C revealed a crossreacting protein of approximately 80 kDa. As the concentration of the protein kinase C activators N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9) or 1-oleoyl-2-acetylglycerol (OAG) was increased, the motility of intact spermatozoa at 30 degrees C was reduced. However, this inhibition of motility was reversed by reducing the concentrations of activators. Even in the presence of 1 mmol CaCl2 l-1, the addition of SC-9 and OAG inhibited the motility of intact spermatozoa. In contrast, the motility of demembranated spermatozoa was not inhibited by the addition of SC-9 or OAG at 30 degrees C. However, the addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a protein kinase C inhibitor, did not appreciably affect the motility of either intact or demembranated spermatozoa at 30 degrees C. At 40 degrees C, both intact and demembranated spermatozoa were almost immotile in the presence or absence of the activators or inhibitor. Intracellular free Ca2+ concentrations, measured by means of a fluorescent Ca2+ indicator, fura-2, gradually increased after the addition of SC-9 and OAG, but no changes were observed in H-7-treated spermatozoa. These results suggest that endogenous protein kinase C is present in the cytoplasmic matrix or the membrane, but is not retained in the axoneme, and that the activation of this enzyme may contribute to a decrease in the flagellar movement of fowl spermatozoa.
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PMID:Possible role of protein kinase C in regulation of flagellar motility and intracellular free Ca2+ concentration of fowl spermatozoa. 796 4

P1 protamines isolated from ejaculated human, stallion, bull, boar and ram spermatozoa and P2 protamines from human and stallion spermatozoa were subjected, after alkaline phosphatase treatment, to in vitro phosphorylation reactions using cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). All P1 protamines were phosphorylated by PKA, whereas P2 protamines were phosphorylated only by PKC. In addition, human, stallion and boar, but not bull and ram, P1 protamines were phosphorylated by PKC. After phosphoamino acid analysis, the protamines showing positive signals for phosphoserine (P-Ser) were subjected to P-Ser conversion reaction and protein sequencing. Only stallion (St1) and human (HP1) P1 protamines contained P-Ser after PKA phosphorylation, located in the middle region of the molecule, i.e., at Ser29 in St1 and Ser28 in HP1. All other phosphorylated P1 protamines contained only P-Thr, which could not be further localized in the sequence with the present methods. After PKC phosphorylation, the internally located Ser residues in human (ser21) and stallion (Ser29) P1 protamines were phosphorylated and, in boar P1 protamine, only Thr43 was slightly phosphorylated. The N-terminally located Ser residues in P1 protamines, which are known to be phosphorylated in vivo, were not phosphorylated by either kinase, indicating that there must still be other types of protamine kinases in sperm cells responsible for their phosphorylation. Within P2 protamines, HP2 was equally well phosphorylated at all Ser residues in addition to some Thr phosphorylation, whereas, in St2, Ser32 was the main target for PKC phosphorylation in vitro. Collectively, PKC is a good candidate for in vivo phosphorylation of P2 protamines and PKA for phosphorylation of some hydroxyamino acid residues in P1 protamines.
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PMID:P2 protamines are phosphorylated in vitro by protein kinase C, whereas P1 protamines prefer cAMP-dependent protein kinase. A comparative study of five mammalian species. 803 90

1. We have investigated the susceptibility of ram sperm phospholipase A2 (PLA2) to stimulation by diacyl- and alkylacylglycerols and by monoacyl- and monoalkylglycerols. 2. PLA2 activity in sonicates from ram spermatozoa was enhanced when 1-stearoyl-2-arachidonoyl-sn-glycerol, the diacylglycerol usually generated by polyphosphoinositide breakdown, was added to a radioactive phosphatidylcholine substrate; the effect was time- and Ca(2+)-dependent. 3. Both diacyl- and alkylacylglycerol considerably enhanced PLA2 activity; 1-O-hexadecyl-2-O-methyl-rac-glycerol, however, only showed slight stimulatory ability. 4. The monoradylglycerols 1-monohexadecanoyl-rac-glycerol, 2-monohexadecanoylglycerol, and 1-O-hexadecyl-sn-glycerol had very little effect on the enzyme's activity. 5. Exposure of spermatozoa to 1-oleoyl-2-acetyl-sn-glycerol (OAG) or 1-O-hexadecyl-2-acetyl-rac-glycerol (1-O-C16/2-C2), when cells were stimulated with the ionophore A23187 and Ca2+, resulted in higher PLA2 activity in sperm sonicates. Furthermore, parallel experiments showed that exocytosis was enhanced if spermatozoa were treated with A23187/Ca2+ and either OAG or 1-O-C16/2-C2. Since both diacyl- and alkylacylglycerols increased PLA2 activity and exocytosis, stimulation of PLA2 activity by these diglycerides may take place independently from protein kinase C activation.
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PMID:Diacylglycerol and alkylacylglycerol stimulate ram sperm phospholipase A2. 806 19

In ram spermatozoa, treatment with the ionophore A23187 and Ca2+ led to an increase in total diacylglycerol mass and to exocytosis of the acrosomal granule. If sperm cells were prelabeled with [3H]palmitic acid, stimulation with A23187/Ca2+ resulted in the generation of [3H]diacylglycerols with a mixture of saturated and unsaturated fatty acids. When cells were prelabeled with 1-O-[3H]octadecylglycerophosphocholine, stimulation led to the generation of [3H]alkylacylglycerol. No rise in [3H]diacyl- or [3H]alkylacylphosphatidic acid was detected under these conditions. Moreover, no changes in the mass of phosphatidic acid have been previously noted under similar conditions. Thus, these results indicate that diradylglycerols are generated via phospholipase C (PLC). Increases in diradylglycerols were paralleled by rises in monoacyl- or monoalkylglycerols labeled at position 1, but not in free [3H]palmitic acid or [3H]octadecanol, implying that, unlike somatic cells, spermatozoa catabolize diradylglycerols via a 2-diglyceride lipase. Activation of PLC appears to be effected by phosphoinositide-derived diacylglycerol: exposure to Mg2+, a cation known to inhibit phosphoinositide hydrolysis, resulted in less PLC activity upon stimulation, and addition of exogenous 1,2-diacylglycerols enhanced the enzyme's activity. However, 1,3-diacylglycerol and alkylacylglycerol also stimulated PLC activity, suggesting that the effect is unlikely to be mediated via protein kinase C. Since diradylglycerols are known to be essential in the molecular sequence leading to membrane fusion in mammalian spermatozoa, these results suggest that their generation via PLC constitutes a fundamental event during acrosomal exocytosis in response to physiological agonists.
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PMID:Polyphosphoinositide-derived diacylglycerol stimulates the hydrolysis of phosphatidylcholine by phospholipase C during exocytosis of the ram sperm acrosome. Effect is not mediated by protein kinase C. 808 26

Acrosomal loss was induced in marsupial spermatozoa by an intermediate of the phosphoinositide pathway. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8; 100 mumol l-1) induced acrosomal loss in 70% of brushtail possum (Trichosurus vulpecula) spermatozoa and in 80% of tammar wallaby (Macropus eugenii) spermatozoa. The DiC8-induced acrosomal loss was not enhanced by co-incubation with calcium ionophore A23187 and occurred in Ca(2+)-free medium and in the presence of the calcium chelator EGTA (3 mmol l-1). There was no evidence of uptake of 45Ca2+ during the DiC8-induced acrosomal loss. Inhibitors of protein kinase C [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] and phospholipase A2 [dexamethasone] did not effect DiC8-induced acrosomal loss in wallaby spermatozoa. The phorbol ester, phorbol 12-myristate 13-acetate, at a concentration of 10 mumol l-1 had no effect on possum spermatozoa and induced acrosomal loss in only 6% of wallaby spermatozoa. It appears that the DiC8-induced acrosome reaction is not mediated by activation of the phosphoinositide pathway and that extracellular calcium is not required for the membrane fusion event. As acrosomal loss was seen only at relatively high concentrations of diacylglycerol (> 50 mumol l-1) and there is no evidence of involvement of other phosphoinositide intermediates or analogues, it is likely that its role is as a direct membrane fusogen.
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PMID:Role of diacylglycerols and calcium in the marsupial acrosome reaction. 810 18


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