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: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two protein kinases active on casein and phosvitin were partially purified from the soluble fraction of ejaculated bovine spermatozoa. They were operationally termed casein kinase A and B based on the order of their elution from a phosphocellulose column. CK-A showed an approximate molecular mass of 38 kDa, and it phosphorylated serine residues of casein and phosvitin utilizing ATP as a phosphate donor (Km 19 microM). Enzyme activity was maximal in the presence of 10 mM MgCl2, whereas it decreased in the presence of spermine, polylysine, quercetin, and NaCl (20-250 mM). CK-B seemed to have a monomeric structure of about 41 kDa; it underwent autophosphorylation and cross-reacted with polyclonal antibodies raised against recombinant alpha, but not beta, subunit of human type 2 casein kinase. It phosphorylated both serine and threonine residues of casein and phosvitin, utilizing ATP (Km 12 microM) but not GTP as a phosphate donor. Threonine was more affected in the phosphorylated phosvitin than in the partially dephosphorylated substrate. CK-B was active toward the synthetic peptide Ser-(Glu)5 and calmodulin (in the latter case, in the presence of polylysine), and it was activated by spermine, polylysine, MgCl2 (30 mM), and NaCl (20-400 mM). The activity of the enzymes was not affected by cAMP, or the heat-stable inhibitor of the cAMP-dependent protein kinase, or calcium.
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PMID:Purification and characterization of two casein kinases from ejaculated bovine spermatozoa. 129 85

Under moderate conditions, reactive oxygen species (ROS) have been shown to inhibit sperm motility after several hours of incubation. The rapid decrease in flagellar beat frequency observed within the first hour of contact between ROS and spermatozoa was associated with a rapid loss of intracellular adenosine triphosphate (ATP). Motility of intact spermatozoa ceased when their ATP concentration was reduced by 85 +/- 5%. Axonemal damage was confirmed when ROS-treated spermatozoa could not reactivate motility after demembranation in a medium containing magnesium adenosine triphosphate (Mg.ATP). However, in conditions allowing rephosphorylation of the axonemes (addition of cyclic adenosine monophosphate, or cAMP, and protein kinase or sperm extracts to the demembranation medium), the motility could reactivate. Three lines of evidence suggested that ATP depletion induced by ROS treatment was responsible for the effects observed in spermatozoa. First, the rapid decrease in intracellular ATP observed after ROS treatment was closely followed by a decrease in beat frequency, loss of intact sperm motility, and axonemal damage due to insufficient phosphorylation. Second, incubation of spermatozoa with the combination pyruvate-lactate allowed maintenance of sperm ATP at a normal level and prevented the effects of ROS; furthermore, spermatozoa immobilized after ROS treatment, then supplemented with pyruvate-lactate, were able to reinitiate motility in parallel with an increase in their ATP level. Third, treatment of spermatozoa with rotenone, an ATP depleting agent, produced effects similar to ROS treatment and could also be reversed by the addition of pyruvate-lactate. These data are consistent with the conclusion that ROS treatment produced axonemal damage mostly as a result of ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility. 133 Oct 7

The acrosome reaction of spermatozoa appears to be analogous to various somatic cell exocytotic events which involve cascade reactions, i.e., transmission of an external signal across the cell membrane resulting in activation of an "amplifier" enzyme and the generation of a second messenger. Using a synchronous acrosome reaction system (De Jonge et al., J. Androl., 10:232-239, '89a), it was found that analogues of the second-messenger cAMP, dibutyryl cAMP (dbcAMP) and 8-bromo cAMP, stimulated the acrosome reaction of capacitated spermatozoa. Additionally, treatment of spermatozoa with either xanthine or non-xanthine phosphodiesterase inhibitors induced a significant (P less than 0.05) increase in the percent acrosome reaction after a period of capacitation in comparison to untreated controls. These results indicate that analogues of cAMP or inhibitors which prevent cAMP hydrolysis can induce the human sperm acrosome reaction. Subsequent experiments were conducted to test whether the amplifier enzyme in the cascade reaction, adenylate cyclase, has a role in the acrosome reaction. Forskolin, an adenylate cyclase stimulator, caused a significant (P less than 0.01) increase in the percent acrosome reaction in comparison to controls. Modulators of adenylate cyclase--adenosine, 2'-0-methyladenosine, and 2',3'-dideoxyadenosine--significantly (P less than 0.01) inhibited the forskolin-induced acrosome reaction. dbcAMP was able to overcome the inhibition by adenosine. Two inhibitors of protein kinase A, the Walsh inhibitor and H-8, caused a significant (P less than 0.01) inhibition of the dbcAMP-induced acrosome reaction. Finally, in the absence of extracellular calcium, dbcAMP induced a significant (P less than 0.01) increase in the acrosome reaction in contrast to A23187. These results suggest that: 1) a molecular mechanism for the human sperm acrosome reaction involves the cAMP second-messenger system; i.e., activation of adenylate cyclase, the amplifier enzyme that produces cAMP, production of cAMP as a second messenger, and activation of cAMP-dependent kinase A; and that 2) activation of adenylate cyclase occurs after calcium influx.
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PMID:Modulation of the human sperm acrosome reaction by effectors of the adenylate cyclase/cyclic AMP second-messenger pathway. 165 65

The cell division cycle in eukaryotes contains up to three major transition points; the conversion of quiescent cells to a stage of active proliferation, the initiation of DNA synthesis (S phase) and the induction of mitosis in cells with newly replicated genome (M phase). Within the past years two strategies, have converged to identify, genetically and biochemically a key protein kinase p34 cdc2 that governs the entry into mitosis. In the fission yeast Schizosaccharomyces pombe a number of mutants in the mitotic regulatory circuit have been isolated. A central gene in the network is cdc2 which is essential for the proper execution of mitosis. The cdc2 gene interacts with a number of other genes for correct mitotic control. The Amphibian oocyte, the oocyte from Xenopus laevis particularly, is arrested at the G2 phase of the first meiotic division; when it enters M phase, it contains a dominant regulatory factor known as MPF (M-phase or maturation promoting factor). Purified MPF is an heterodimer formed of two polypeptides p34cdc2 an homologue of the product of the gene cdc2 and p45cdc13 or cyclin an homologue of the product of the gene cdc13. Biochemical studies have revealed that p34cdc2 is a phosphotyrosine protein during the G2 phase of the cell cycle, both mitotic and meiotic. The tyrosine phosphorylation of p34cdc2 is regulated by the gradual accumulation of cyclin. At the onset of M phase, the complex p34cdc2/cyclin is activated as an histone H1 kinase, and p34cdc2 is tyrosine dephosphorylated. The mechanism of activation of p34cdc2 is negatively regulated by a form of protein phosphatase 2A. Ovulated vertebrate oocytes are arrested at metaphase of the second meiotic division (M II) under the control of the proto-oncogene c-mos a protein kinase. The exit of M II phase and the initiation of early embryonic mitotic cell cycles are physiologically induced by the spermatozoa at the time of fertilization. They requires the degradation of c-mos by a Ca2+ dependent proteolytic enzyme and the destruction of cyclin by an ubiquitin dependent pathway. The Xenopus oocyte has led to the molecular elucidation of MPF and identified links between cell cycle control, protein phosphorylation and proto-oncogenes. Despite the impresive progess of recent years, there is still much to be learned about the control of meiosis in Xenopus oocytes.
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PMID:[From ovocyte to biochemistry of the cell cycle]. 165 57

A high molecular mass dynein ATPase polypeptide and a 18-20 kDa dynein light chain of Ciona sperm flagella are phosphorylated during in vivo activation of motility or in vitro activation of motility by incubation with cyclic AMP. A similar level of phosphorylation of these proteins is obtained by incubation of washed, demembranated spermatozoa with catalytic subunit of cyclic AMP-dependent protein kinase, under conditions where there is no activation of motility until a supernatant component is added. Therefore, phosphorylation of these dynein polypeptides is not sufficient for activation of motility. Activation of motility in vitro by incubation with cyclic AMP can be completely inhibited by a random copolymer of glutamate and tyrosine that inhibits tyrosine kinase activity. Under these conditions, much of the protein phosphorylation associated with activation of motility is also inhibited. These new results suggest that regulation of motility of these spermatozoa may involve a multicomponent kinase cascade rather than a simple phosphorylation of a protein 'switch' by the cyclic AMP-dependent kinase. A 53 kDa axonemal phosphoprotein band, identified as band M1, shows the strongest correlation with activation of motility in these experiments.
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PMID:Activation of Ciona sperm motility: phosphorylation of dynein polypeptides and effects of a tyrosine kinase inhibitor. 166 61

A protein-tyrosine kinase has been isolated from a detergent-soluble extract of boar spermatozoa, using poly(Glu, Tyr)4:1 as a substrate. The purification procedure involves sequential column chromatographies on phosphocellulose, polyamino acid affinity and Sephadex G-100 molecular sieving, and results in more than a 1200-fold enrichment. Analysis of the most purified preparation by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a major Coomassie blue-stained band of molecular mass 42 kDa. The Tyr-protein kinase does not seem to be autophosphorylable. The Km value for poly(Glu, Tyr)4:1 is relatively low, 2.3 microM, and the tyrosine-polymer phosphorylating activity is apparently inhibited by tyrphostin. The characteristics shown by this new tyrosine kinase--the first to be described in mature male germ cells--support the hypothesis that it belongs to the group of non-receptor-associated tyrosine kinases.
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PMID:Tyrosine protein kinase in boar spermatozoa: identification and partial characterization. 173 32

Intact cauda-epididymal mature and caput-epididymal immature goat spermatozoa were assessed for their capacity to phosphorylate the outer surface proteins upon incubation in a modified Ringer's solution containing [delta-32P]ATP. The immature spermatozoa possessed markedly greater (approximately 7-fold) efficacy to phosphorylate the ecto-proteins than the mature cells. Autoradiographic analysis of the 32P-labelled proteins resolved by SDS-PAGE, showed that multiple sperm ecto-proteins are phosphorylated by an endogenous ecto-cyclic AMP-independent protein kinase (CIK) and the phosphorylation profile of these proteins underwent marked alteration during the epididymal sperm maturation. The intact caput-sperm as well showed nearly 4-fold higher specific activity of ecto-CIK than the cauda-sperm when the kinase activity was estimated using phosvitin as the exogenous protein substrate. The data suggest that the ecto-CIK and its specific protein substrates located on the sperm outer-surface, may have important roles in regulating the epididymal maturation of the male gametes.
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PMID:Alteration of the ecto-protein phosphorylation profile of intact goat spermatozoa during epididymal maturation. 187 97

Casein kinase II from bovine epididymal spermatozoa was purified to apparent homogeneity by repeated chromatography with phosphocellulose and gel filtration with sephacryl S-200. The purified enzyme exhibited a molecular mass of 130 kDa by gel filtration and displayed three polypeptide bands with molecular masses of 26, 33, and 36 kDa by SDS-polyacrylamide gel electrophoresis. Antibodies raised against calf thymus casein kinase II cross reacted with the three sperm polypeptides. Incubation of the holoenzyme with either [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of the 26-kDa subunit. The enzymatic activity with casein as substrate was strongly inhibited by nanomolar heparin and greatly stimulated by micromolar spermine. With casein as substrate, the specific activity of the pure enzyme (0.5 mumol/min/mg protein) was comparable to that of casein kinase II from other sources. Endogenous substrates of the kinase were demonstrated by incubating sperm cytosolic extracts with [gamma-32P]GTP, under conditions that limit the expression of other protein kinases, and analyzing the products by SDS-PAGE and autoradiography. Similar results were obtained when sperm extracts, suitably diluted to minimize endogenous casein kinase II, were incubated with [gamma-32P]GTP and aliquots of pure sperm casein kinase II. Low concentrations (50 microM) spermine strongly enhanced the phosphorylation of 92- and 106-kDa cytosolic proteins. Our results clearly show that casein kinase II is present in spermatozoa and that it shares many of the properties of the enzyme from other sources. Further, they indicate that the enzyme plays a role in mediating the phosphorylation state of sperm proteins.
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PMID:Purification and characterization of polyamine-stimulated protein kinase (casein kinase II) from bovine spermatozoa. 189 32

Several lines of evidence have demonstrated conclusively the presence of cAMP-dependent protein kinase (ecto-RC) activity on the external surface of goat cauda-epididymal intact spermatozoa. The intact-cell ecto-kinase that caused transfer of the terminal phosphate of exogenous ATP to the serine and threonine residues of exogenous histone was specifically activated by cAMP. As well, the ecto-kinase caused phosphorylation of the synthetic peptide Kemptide. The isolated spermatozoa, before or after incubation with reaction mixture for ecto-kinase assays, were approximately 99.5% viable, as shown by the analyses of ethidium bromide fluorescence and the cytosolic marker enzymes lactic dehydrogenase and 3-phosphoglycerate kinase. The ecto-kinase activity was not due to contamination of epididymal plasma and damaged cells or to protein kinase that may have leaked from the cells. There was little uptake of ATP and histone by the cells. The intact-cell kinase activity was strongly (80-90%) inhibited by treatment with membrane nonpenetrating surface probes: p-chloromercuriphenylsulfonic acid (2 microM), diazonium salt of sulfanilic acid (DSS, 0.5 mM), and proteases such as trypsin, chymotrypsin, and pronase (each 125 micrograms/mL). Disruption of sperm plasma membrane by sonication or Triton X-100 (0.2%) caused about a fivefold increase of the intact sperm kinase activity. Highly purified sperm plasma membrane (PM) possessed ecto-kinase activity that was resolved into type I and II kinases by DEAE-cellulose chromatography, the type I isoenzyme being the major (approximately 70%) enzymic species. Treatment of the intact spermatozoa with DSS prior to isolation of PM caused a marked loss of the activities of both the isoenzymes, indicating thereby the "ecto" nature of the PM-bound type I and II kinases. Preparations of vigorously forward-motile spermatozoa with 100% intactness had approximately fourfold higher specific activity of the ecto-kinase than the "composite" cells from which the former cells were isolated. However, the profiles of the type I and II ecto-kinases of the composite, as well as forward-motile spermatozoa, were nearly identical. The data are consistent with the view that ecto-kinases may have role in the regulation of flagellar motility.
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PMID:Type I and II cAMP-dependent ecto-protein kinases in goat epididymal spermatozoa and their enriched activities in forward-motile spermatozoa. 216 Aug 33

Studies of in vitro models demonstrate that a forward motility protein (FMP) is required for the initiation of forward motility in the immature epididymal spermatozoa. FMP is a heat-stable glycoprotein derived from epididymal plasma. During the epididymal maturation of spermatozoa in vivo, there is a marked increase of intrasperm pH and level of cyclic adenosine monophosphate (cAMP). Several studies suggest that exogenous FMP in concert with elevated intrasperm pH and level of cAMP initiates flagellar motility during the epididymal transit of sperm. cAMP activates sperm cytosolic cAMP-dependent protein kinases, which in turn phosphorylate multiple intrasperm phosphoproteins that may regulate flagellar motility. Exogenous calcium ion activates intact sperm motility, although it inhibits motility of demembranated cells on reactivation. Occurrence of cAMP-dependent type I and II protein kinases, a novel cAMP-independent protein kinase, and a phosphoprotein phosphatase has been demonstrated on the external surface of spermatozoa. The sperm surface has a coupled-enzyme system: ecto-cAMP-independent protein kinase and phosphoprotein phosphatase that regulate the phosphorylation and dephosphorylation of endogenous sperm ectophosphoproteins. The specific activities of these ecto-enzymes increase markedly during forward progression, suggesting that they may have a role in regulating flagellar motility.
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PMID:Biochemical parameters of initiation and regulation of sperm motility. 219 32


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