Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 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

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

When sea urchin spermatozoa were treated with a Triton X-100 solution, cAMP-dependent protein kinase (cA-kinase) activity was extracted. Further extraction with Triton X-100 of axonemes isolated from the Triton-extracted sperm again released a considerable amount of the cA-kinase activity. The activity which remained after extraction three times with Triton X-100 was released by treatment with a low salt solution. These activities found in the various extracts were likely to be due to the same cA-kinase, which was a mammalian type II-like enzyme. The cA-kinase activity that remained in the axonemes after the first Triton X-100 extraction may be involved in the regulation of flagellar movement in the Triton-extracted sperm.
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PMID:The cAMP-dependent protein kinase in sea urchin sperm tails: association of the enzyme with the flagellar axonemes. 222 2

Mammalian spermatozoa have been shown to possess cAMP-dependent protein kinase (A-PK) and endogenous substrate proteins for this enzyme. A study of the kinase system was undertaken to determine changes that may be associated with sperm maturation by comparing immature testicular with mature cauda epididymal and ejaculated spermatozoa. Absolute activity levels of A-PK, stimulated over a concentration range of 10(-9) to 10(-5) M, was significantly greater in testicular than ejaculated spermatozoa. At an optimal cAMP concentration (10(-6) M), testicular spermatozoa had significantly greater amounts of cAMP-dependent protein kinase activity than did cauda or ejaculated spermatozoa. Electrophoretic analysis and autoradiography of NP-40-soluble protein extracts revealed the presence of two substrate proteins (Mr = 62,000 and 44,000) in all three types of spermatozoa. In addition, a phosphoprotein (Mr = 20,000) was detected in mature cauda and ejaculated but not immature testicular spermatozoa. The phosphorylation of these substrate proteins was both dose and time dependent. Examination of cyclic AMP phosphodiesterase activity revealed significantly higher levels in testicular than ejaculated spermatozoa. These results indicate marked alterations in cAMP-modulated protein phosphorylation and dephosphorylation systems in ram spermatozoa during epididymal maturation.
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PMID:Characterization of cAMP-dependent protein kinase and its endogenous substrate proteins in ram testicular, cauda epididymal, and ejaculated spermatozoa. 285 34

Calmodulin concentration and cAMP-dependent protein kinase activity were simultaneously determined on ram spermatozoa collected by cannulation of successive segments of the epididymal tubule. Epididymal transit was characterized on one hand by an overall decrease in the calmodulin level and on the other by a dramatic rise in the cAMP-dependent protein kinase activity. In contrast to the calmodulin level, the cAMP-dependent protein kinase activity was correlated with the acquisition of flagellar beat. No further alterations in the level of these two proteins could be detected as spermatozoa acquired progressive motility.
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PMID:Changes in calmodulin level and cAMP-dependent protein kinase activity during epididymal maturation of ram spermatozoa. 299 10

8-Azido cAMP photoaffinity labeling of cAMP-dependent protein kinase regulatory subunits (R1 = 49 K;R2 = 55K) was done on spermatozoa from species lacking, and species containing an epididymis. Spermatozoa from sea urchin and trout contained only R1, while rat caudaepididymal spermatozoa contained both R1 and R2 subunits. This was established by the Mr value of the 8-azido cAMP photolabeled moieties, and a biochemical analysis based on the known differences of protein-nucleotide interactions of Type I and II cAMP-dependent protein kinases. Sea urchin and trout sperm R1 subunits were similar to mammalian sperm R1 subunits in co-migration on SDS-polyacrylamide gels and in both saturation and specificity of nucleotide binding. Calcium enhanced photoprobe binding to rat R1 and R2 subunits and to sea urchin R1 subunit without revealing a sea urchin R2 subunit. Likewise, phosphodiesterase incubation of sea urchin and trout spermatozoa prior to photolabeling did not reveal R2 subunits. These data suggest that the cAMP regulation of sperm physiology may require R1 subunit in species both with and without an epididymis. Further taxonomic study is necessary to determine whether evolutionary acquisition of the epididymis and internal fertilization may have created unique environments favoring the addition of sperm R2 regulatory subunits of cAMP-dependent protein kinase.
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PMID:A comparative analysis of cAMP-dependent protein kinase regulatory subunits in sea urchin and rat spermatozoa. 299 17

By using ethidium bromide fluorescence to measure cellular permeability and the photoaffinity probe, 8-azido-[32P] cyclic adenosine monophosphate (cAMP), to label cAMP-dependent protein kinases, washed bovine epididymal spermatozoa were examined for the presence of "ectokinases" on the sperm surface. In washed, intact spermatozoa, three proteins of Mr 49,000, 54,000, and 56,000 specifically bound 8-azido-[32P] cAMP. The Mr 49,000 protein corresponded to the type I regulatory subunit while the Mr 56,000 and 54,000 proteins comigrated with phosphorylated and dephosphorylated forms, respectively, of type IIA regulatory subunit of bovine heart. The addition of Nonidet P-40 (0.1%) increased the radioactive labeling of all three proteins and caused the appearance of a cAMP binding protein of Mr 40,000, which was likely a proteolytic fragment of the regulatory subunit. Although these data could support the concept of a surface location for regulatory subunits in spermatozoa, it was necessary to determine if the appearance of cAMP binding sites was correlated with the loss of membrane integrity. A population of washed epididymal spermatozoa appeared to contain 10-20% damaged cells based on ethidium bromide fluorescence. The same population of cells also had 10-20% of the regulatory subunits of the cAMP-dependent protein kinase accessible to labeling with the cyclic AMP photoaffinity probe. When spermatozoa were sonicated for increasing lengths of time, ethidium bromide fluorescence was found to be related directly to the relative amount of regulatory subunit labeling by the probe. It is suggested that the major apparent cAMP-dependent "ectokinases" in sperm represent artifacts resulting from cellular damage.
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PMID:Cyclic AMP-dependent protein kinase isozymes of bovine epididymal spermatozoa: evidence against the existence of an ectokinase. 301 Nov 34

The creation of an environment in mouse fallopian tubes that is sufficient to sustain preimplantation embryo development is known to require the participation of spermatozoa in excess of those involved in the process of fertilization. We have now found that highly purified cAMP-dependent protein kinase can substitute for spermatozoa in the facilitation of the first cleavage of mouse embryos. Both spermatozoa and purified protein kinase induce increases in fallopian phosphoproteins. It is suggested that nonfertilizing spermatozoa could exert their effects on preimplantation embryo development through the provision of protein kinase.
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PMID:Reproduction in mice: protein kinase mimics the sperm effect on preimplantation embryo development. 350 8

A heat-stable microtubule-associated protein (MAP) with molecular weight of 190,000, termed 190-kD MAP, was purified from bovine adrenal cortex. This MAP showed the same level of ability to promote tubulin polymerization as did MAP2 and tau from mammalian brains. Relatively high amounts of 190-kD MAP could bind to microtubules reconstituted in the presence of taxol. At maximum 1 mol of 190-kD MAP could bind to 2.3 mol of tubulin. 190-kD MAP was phosphorylated by a cAMP-dependent protein kinase prepared from sea urchin spermatozoa and by protein kinase(s) present in the microtubule protein fraction prepared from mammalian brains. The maximal numbers of incorporated phosphate were approximately 0.2 and approximately 0.4 mol per mole of 190-kD MAP, respectively. These values were lower than that of MAP2, which could be heavily phosphorylated by the endogenous protein kinase(s) up to 5 mol per mole of MAP2 under the same assay condition. 190-kD MAP had no effects on the low-shear viscosity of actin and did not induce an increase in turbidity of the actin solution. It was also revealed that 190-kD MAP does not cosediment with actin filaments. These data clearly show that, distinct from MAP2 and tau, this MAP does not interact with actin. Electron microscopic observation of the rotary-shadowed images of 190-kD MAP showed the molecular shape to be a long, thin, flexible rod with a contour length of approximately 100 nm. Quick-freeze, deep-etch replicas of the microtubules reconstituted from 190-kD MAP and brain tubulin revealed many cross-bridges connecting microtubules with each other.
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PMID:Purification and characterization of a 190-kD microtubule-associated protein from bovine adrenal cortex. 378 89

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


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