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)

Goat cauda-epididymal intact spermatozoa have been shown to possess an ecto-cyclic AMP-independent protein kinase activity on the external surface that causes phosphorylation of the serine and threonine residues of exogenous phosvitin. The enzyme is neither a tyrosine kinase nor a catalytic subunit of the cyclic AMP-dependent protein kinase. It is not activated by Ca2+, calmodulin and phosphatidylserine. The intact-cell enzyme is capable of phosphorylating a variety of proteins including sperm plasma membrane-bound phosphoprotein(s). The enzymic activity of the intact spermatozoa was not due to contamination of broken or "leaky" cells. The kinase activity of the whole cells was strongly inhibited by the non-penetrating surface probes: p-chloromercuriphenylsulphonic acid (10 microM) and proteases (125 micrograms/ml). The specific activity of the ecto-kinase increased nearly 100% during vigorous forward progression of spermatozoa.
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PMID:An ecto-cyclic AMP-independent protein kinase in goat spermatozoa and its change of activity during forward motility. 381 58

The changes in intracellular calmodulin levels and cAMP-dependent protein kinase activities have been studied in the testis of normally developing and hypophysectomized rats. It appears that the onset of spermatogenesis which occurs on the first days of the postnatal development, is associated with a major (over fivefold) increase in the calmodulin level and enhancement of the cAMP-dependent protein kinase activity. On the contrary, hypophysectomy of adult animals is associated with a progressive decline in the calmodulin level and a rapid and regular decrease in the cAMP-dependent protein kinase activity. Moreover, measurements of the intracellular calmodulin level and cAMP-dependent kinase activity of isolated testicular germ cells or epididymal spermatozoa have shown that testosterone, administered to hypophysectomized rats as subcutaneous implants, maintains the concentration of these regulatory proteins to normal values.
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PMID:Calmodulin level and cAMP-dependent protein kinase activity in rat spermatogenic cells and hormonal control of spermatogenesis. 397 73

The acrosomal membrane of mammalian spermatozoa is segregated into domains of different structure and function. The outer acrosomal membrane of the apical and principal segments is the only domain to participate in the membrane fusion events of the acrosome reaction, but the molecular basis for this function is not resolved. In previous studies of bovine spermatozoa, we noted that a unique structural feature of the outer acrosomal membrane was an adherent layer of electron-dense material on its luminal surface (ES Surface, Branton et al., 1975). In this study, we report the isolation of this material and we describe both its structural and biochemical characteristics. Cauda epididymal spermatozoa were extracted with 1% Triton X-100 to solubilize cytoplasmic and membrane components; detergent treatment solubilized the outer acrosomal membrane but not its adherent electron-dense complex. Homogenization released this complex from the spermatozoa and it was then resolved into a homogeneous fraction by centrifugation on Percoll density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction revealed a spectrum of polypeptides including components of 290 kDa, 280 kDa, 260 kDa, 115 kDa, 81 kDa, 58 kDa, and 46 kDa and a family of interrelated components in the 34-12 kDa range. This complex possesses protein kinase activity that phosphorylates specific endogeneous polypeptides in a cAMP-independent manner. In addition, several polypeptides of the 34-12 kDa family specifically bind 125I-calmodulin. One consistent structural response of the isolated complex was that its edges wound into a spiral configuration. We speculate that this membrane-associated assembly plays a functional role in the membrane fusion events of the acrosome reaction.
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PMID:Isolation and characterization of a macromolecular complex associated with the outer acrosomal membrane of bovine spermatozoa. 405 33

Plasma membranes from bovine epididymal spermatozoa possess both cAMP-independent and cAMP-dependent protein kinase activity. With the synthetic peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly as substrate, the basal activity of the membrane-associated protein kinase(s) was 0.1 nmol phosphate incorporated X min X mg protein. In the presence of 5 microM cAMP, the apparent activity was increased about twofold. The addition of Nonidet P-40 (0.05%) to the assay mixture increased protein kinase activity to 0.4 and 4.0 nmol phosphate incorporated X min X mg protein in the absence or presence of 5 microM cAMP, respectively. Both isozymes of the cAMP-dependent protein kinase were detected in detergent-solubilized membranes but 95% of the activity appeared as a Type II form based on DEAE-Sephacel chromatography. Several polypeptide components of the plasma membrane served as substrates for membrane-associated cAMP-dependent protein kinases, in vitro. In the absence of detergent, two cAMP-dependent phosphoproteins of 41,000 Mr and 60,000 Mr were detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. When 0.05% Nonidet P-40 was included in the assay mixture, a cAMP-dependent phosphoprotein of 43,000 Mr appeared. Two-dimensional polyacrylamide gel electrophoresis of membranes phosphorylated in the presence of 5 microM and 0.05% Nonidet P-40 revealed phosphoproteins of the following molecular weights/isoelectric points: 56,000/6.7, 56,000/6.9, 51,000/6.2, 42,000/5.9, 42,000/6.0, 38,000/6.1, 38,000/6.4, 14,000/7.2, 12,000/7.4 and a train of five polypeptides appearing at 14,000/5.4-6.0.
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PMID:Protein phosphorylation of plasma membranes from bovine epididymal spermatozoa. 608 45

Cycl AMP concentrations were elevated and acrosome reactions were induced in intact sea urchin spermatozoa by Nigericin, A23187, and pH 9.0 seawater. To determine whether or not the metabolism of cyclic AMP was being altered in sperm heads, the heads were mechanically separated from the flagella, and the flagella-less heads were then isolated by differential centrifugation. The isolated heads contained 1 to 2 nmol of ATP and 1 to 2 pmol of cyclic AMP/mg wet weight and retained these concentrations for several hours if stored at 0 degrees C. The flagella-less heads also retained the mitochondria of the midpiece area. The heads retained their functional status and could be stimulated to undergo acrosome reactions (filament extension) in response to Nigericin, A23187, or pH 9.0 seawater. Furthermore, the isolated heads could activate sea urchin eggs after induction of an acrosome reaction by Nigericin or pH 9.0 seawater. The isolated heads contained appreciable adenylate cyclase, cyclic AMP phosphodiesterase, cyclic GMP phosphodiesterase, guanylate cyclase, cyclic AMP-dependent protein kinase, and calmodulin. Nigericin, pH 9.0 seawater, and A23187 caused not only the induction of an acrosome reaction but also elevations of cyclic AMP in the isolated heads, and extracellular Ca2+ was an absolute requirement for both responses. At 16 degrees C, Nigericin caused elevations of cyclic AMP within 5 s, but maximal elevations were not observed until 1 min; it induced a maximal percentage of acrosome reactions by 40 s. Incubation of cells at 0 degrees C resulted in a delay of maximal acrosome reactions until between 10 and 20 min after addition of Nigericin. Under these conditions, maximal elevations of cyclic AMP were observed by 5 min, demonstrating that cyclic AMP elevations precede the complete morphological change associated with an acrosome reaction. ATP concentrations within the sperm heads declined in response to Nigericin, pH 9.0 seawater, or A23187, and its decrease also required the presence of extracellular Ca2+. The decline in ATP concentrations was slightly more rapid in the presence of rotenone, suggestive of some ATP synthetic capabilities of the isolated head preparation. 45Ca2+ uptake was increased by Nigericin elevated pH, and A23187 but was not appreciably altered by monensin. Monensin also did not cause appreciable elevations of cyclic AMP concentrations, induction of an acrosome reaction, or decreases of ATP concentrations. Here, we describe for the first time that cyclic AMP concentrations can be increased in flagella-less heads of spermatozoa and show that these changes are associated with an acrosome reaction.
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PMID:The elevation of cyclic AMP concentrations in flagella-less sea urchin sperm heads. 625 63

The binding properties of cyclic-AMP to intact and fractionated in heads and tails human spermatozoa have been studied. Whole spermatozoa bound 6.8 +/- 0.8 pmol of cyclic-AMP per 10(7) cells. Binding of cyclic-AMP was selectively located in the midpiece-tail region of the spermatozoa. Thus only 18% of the binding in the whole sperm could be accounted for by the binding on the sperm heads. This distribution is similar to that previously described for the protein kinase of bovine sperm. Blocking membrane sulfhydryl groups with pCMBS induced a drastic inhibition (65%) of the cyclic-AMP binding to the sperm heads, while only moderately reducing (30%) the binding in the sperm tails
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PMID:Differential distribution of cyclic-AMP receptors in human spermatozoa. 626 2

Intact spermatozoa from rat cauda epididymides possess an ecto-(cyclic AMP-dependent protein kinase) activity that causes the transfer of the terminal phosphate group of ATP to the serine residues of all the histone fractions. The enzyme showed a high degree of substrate specificity for the phosphorylation of histones rather than protamine, casein and phosvitin. The cell-external-surface protein kinase requires Mg2+ for activity, and other bivalent cations such as Mn2+ and Co2+ can substitute partially for Mg2+, whereas Ca2+ and Zn2+ are potent inhibitors of the enzyme. The enzyme has markedly higher affinity for cyclic AMP than for other cyclic nucleotides for its activation, with an apparent Km value for cyclic AmP of 80 nM. Spermatozoal ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. There was no loss of kinase activity from the cells when washed with 2 mM-EDTA, and the histones phosphorylated by intact spermatozoa were located outside the cells. Protein kinase activity of intact cells was strongly inhibited (approx. 90%) by p-chloromercuribenzenesulphonic acid (10 microM), which is believed not to enter the cells. These data provide further support for the localization of a protein kinase on the external surface of spermatozoa.
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PMID:Enzymic characteristics of an ecto-cyclic AMP-dependent protein kinase in rat epididymal spermatozoa. 627 41

A fraction obtained from detergent-extract of sea urchin or starfish spermatozoa using DEAE-cellulose chromatography reactivated Triton X-100 models of the spermatozoa in a cAMP-dependent manner. The DEAE fraction contained cAMP-dependent protein kinase with a high level of specific activity. Rabbit muscle inhibitor protein highly specific for cAMP-dependent protein kinases inhibited the ability of the deae fraction to induce reactivation of Triton X-100 models.l This inhibition paralleled inhibition of cAMP-dependent protein kinase activity of the DEAE fraction, suggesting participation of the enzyme in the cAMP-dependent reactivation of Triton X-100 models. However, cAMP-dependent protein kinase further purified from the DEAE fraction was incapable of reactivating these models by itself. A protein factor which was separated from the protein kinase in the course of purification of the enzyme was found to also be necessary for the reactivation. When cAMP-dependent protein kinase was pretreated with protein kinase inhibitor before addition of the protein factor, the reactivation of Triton X-100 models was no longer detected. However, after the protein factor had been incubated with cAMP and cAMP-dependent protein kinase, protein kinase inhibitor did not repress reactivation of Triton X-100 models. We propose that the reactivation needs phosphorylation of the protein factor by cAMP-dependent protein kinase.
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PMID:Evidence that cAMP-dependent protein kinase and a protein factor are involved in reactivation of triton X-100 models of sea urchin and starfish spermatozoa. 628 92

Calmodulin level and cAMP-dependent protein kinase activity of ram germ cells at different stages of spermatogenesis have been determined. Calmodulin levels decrease during maturation. Simultaneously, calmodulin localization changes during cell differentiation. In round, elongating, and elongated spermatids, calmodulin is closely associated with the developing acrosome; in spermatozoa, it becomes present in the postacrosome, the neck region and the tail. Protein kinase activity is relatively low in testicular cells but increases dramatically during epididymal maturation of spermatozoa. A concerted regulation by cAMP and Ca2+ of biochemical events in spermatogenic cells and spermatozoa is suggested.
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PMID:Evolution of Ca2+- and cAMP-dependent regulatory mechanisms during ram spermatogenesis. 631 46

The presence and localization in pig and rat testes of phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) and its substrates were investigated. PL-Ca-PK activity was found in the testis total particulate fraction and epididymal fluid, but little in the testis total particulate fraction and epididymal fluid, but little in the testis cytosol and matured spermatozoa obtained from the epididymis. Similarly, at least three endogenous substrates (83,000, 33,000, and 26,000) and five substrates (greater than 100,000, 83,000, 60,000, 43,000, and 19,000) for PL-Ca-PK were detected in the testis total particulate fraction and the epididymal fluid, respectively, but little or no substrates were observed in the testis cytosol and matured spermatozoa. The three substrates detected in the total particulate fraction were also observed in the testis nuclear fraction. In rat testis, PL-Ca-PK activity was detected in the total particulate fraction of germ cells. The results suggested that PL-Ca-PK system might be important in membrane- or subcellular organellar-associated functions in testis.
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PMID:Phospholipid-sensitive Ca2+-dependent protein kinase system in testis: localization and endogenous substrates. 649 74


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