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

Preincubation of isolated epididymal fat cells with dexamethasone or treating rats with cortisol enhances the epinephrine-stimulated lipolysis of the cells as well as cAMP-dependent protein kinase activity in homogenates of these fat cell suspensions. During maximal inhibition of phosphodiesterase activity by theophylline or dibutyryl cAMP, this potentiating effect of glucocorticoids on the fat cells was also present. There was no lowering of the total phosphodiesterase activity in homogenates of fat cell suspensions of rats that were treated with cortisol, but there appeared to be a lower activity of the low KM phosphodiesterase activity. It is concluded that induction of protein kinase by glucocorticoid hormone is responsible for its special type of stimulative action on lipolysis.
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PMID:The mechanism of the potentiating effect of glucocorticoids on catecholamine-induced lipolysis. 16 65

There appear to be two classes of protein kinases in rat heart and adipose tissue, types I and II. Type I elutes from DEAE-cellulose at smaller than 0.1 M NaCl and type II at greater than 0.1 M NaCl. The type I enzyme is more readily dissociated by salt or histone than is the type II enzyme. If the type I kinase is first dissociated by cAMP, the subunits reassociate very slowly at 0 degrees C on removal of the cAMP by Sephadex G-25 chromatography, whereas those of type II reassociate very rapidly. Rat heart contains mostly type I and a small amount of type II enzyme, whereas adipose tissue contains almost exclusively the type II enzyme. The adipose tissue enzyme resembles the heart type II kinase in all of the above properties, although the two enzymes are not identical as indicated by slight differences in elution patterns from DEAE-cellulose columns. Incubation of rat epididymal adipose tissue with low concentrations of epinephrine (0.11 muM) increases glycerol production and the fraction of the protein kinase in the active form (activity ratio). The change in cAMP under these conditions is not statistically significant. The presence of insulin inhibits the epinephrine effect on glycerol production and protein kinase but has no measurable effect on cAMP levels. Incubation of adipose tissue with high epinephrine concentrations (11 muM) increases the cAMP level, the protein kinase activity ratio, and glycerol production. Under these conditions insulin decreases the cAMP level and kinase activity ratio but does not reduce glycerol production. The data suggest that very small changes in the tissue cAMP level, undetectable by the assay method, are magnified during the stepwise activation of glycerol output aided possibly by cooperative effects between cAMP and protein kinase. The procedure developed for determining the state of activation of the cAMP-dependent protein kinase in adipose tissue must be modified by reducing the salt concentration of the buffers in order to carry out similar studies in the heart. This reflects the different types of protein kinase in the two tissues. The addition of charcoal to crude extracts of heart prevents protein kinase activation by added cyclic AMP. Charcoal should therefore prevent any activation that could occur if any sequestered cAMP were released during homogenization. Charcoal addition thereby provides a means to distinguish intracellular cAMP activation of the kinase from that which might occur following cell rupture. If epinephrine-perfused hearts are homogenized in the presence of charcoal, epinephrine stimulation of the protein kinase is only slightly decreased. This indicates that the protein kinase is activated intracellularly by cAMP and suggests that all of the cAMP in the cell is available to the protein kinase; i.e., cAMP is not released during homogenization.
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PMID:Hormonal regulation of adenosine 3',5'-monophosphate-dependent protein kinase. 16 70

Measurements of tissue cyclic AMP (cAMP) concentration, the activity of cAMP-dependent protein kinase and the level of the enzyme's thermostable, macromolecular inhibitor were made on preparations of rat epididymal fat pad from animals fed high fat or high carbohydrate diets. The cAMP concentration from rats adapted to a high lard diet for 14-15 days was 153 +/- 17.8 pmoles/mg protein as opposed to 76 +/- 6.0 found with high glucose diet. No significant difference in total cAMP-dependent protein kinase activity was observed among rats fed high glucose, high lard or laboratory chow, although the enzyme's activity ratio (-cAMP)(+cAMP) was significantly elevated with lard feeding (0.49 +/- 0.02) as opposed to glucose feeding (0.43 +/- 0.01). Crude preparations from lard and glucose fed animals were equivalent in inhibitory activity when tested with enzyme from chow fed animals. Agarose column chromatography separated holoenzyme and C subunit forms of the protein kinase when 500 mM NaCl was present in the elution buffer. Absence of the salt allowed subunit reassociation to occur. Direct addition of NaCl greater than or equal to 75 mM significantly inhibited protein kinase activity. The results indicate that the adipose tissue of rats fed a high lard diet has a higher concentration of cAMP and an increased protein kinase activity ratio than tissue from rats fed a fat free, high glucose diet. Total cAMP-dependent protein kinase activity and the level of a thermostable macromolecular inhibitor remained unchanged.
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PMID:The concentration of cyclic AMP and the activity of cyclic AMP dependent protein kinase and an inhibitor in the adipose tissue of rats fed lard or glucose diets. 21 69

The interaction of rat cauda epididymal sperm cAMP-dependent protein kinase (PKA) with seminal vesicle fluid (SVF) proteins was examined. Specific proteins in SVF act as substrates for the sperm cell PKA. The apparent molecular weights of these proteins are 45.0, 31.5, 17.2, 14.7, and 13.3 kDa. The phosphorylation of one low-molecular-weight cauda sperm protein is blocked in the presence of SVF. There is no PKA enzyme activity in SVF. The presence of phosphate transfer activity between the sperm cell enzyme and the SVF proteins is species dependent. For example, mouse and rat SVF proteins are efficient phosphate acceptors, but there is no phosphorylation activity when hamster SVF is used as the enzyme substrate. The sperm cell samples were also assessed for membrane integrity. Specifically, cauda sperm cells used in these assays were judged to be intact when examined microscopically using the fluorescent vital dye carboxyfluorodiacetate. Although there was enzyme activity in the supernatants of the rat sperm cell samples, in the protein kinase assay it required three times as much supernatant volume (compared with intact cell sample volume) to measure the activity. Supernatant enzyme activity did not increase with washing, indicating that the cells were not damaged by this procedure. The enzyme itself does not adhere to the sperm cells, so the PKA enzyme activity is most likely oriented on the external surface of the sperm cell.
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PMID:Cauda epididymal sperm interactions with seminal vesicle fluid. 151 Aug 46

The lipid fraction ("fat cake") of rat epididymal adipocytes contains a prominent phosphoprotein (62 kDaapp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) that is multiply phosphorylated by cAMP-dependent protein kinase in vivo, at which point it migrates as a 65/67-kDaapp doublet by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is by far the most heavily radiolabeled protein in the cell. Western blot analysis of various tissues with immunopurified antibodies purified from antisera raised against the 62-kDa species suggests that the protein is specific for adipocytes. This protein, which we term perilipin, is found in differentiated cultured 3T3-L1 adipocytes, but not in their precursor 3T3-L1 fibroblasts. Immunocytochemical studies with specific antiserum shows that the perilipin is closely associated with the periphery of lipid storage droplets in cultured adipocytes. Given its adipocyte specificity, acute regulation by hormones, and subcellular location, we speculate that perilipin plays a role in the specialized lipid storage function of adipocytes.
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PMID:Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets. 204 Jun 38

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

The expression of mRNAs for the RI alpha, RII alpha, and C alpha subunits of cAMP-dependent protein kinase has been studied in different ram germ cells. The sizes of the specific RI alpha, RII alpha, and C alpha mRNAs, observed in germ cells were 1.6, 2.0, and 2.6 kb, respectively. RI alpha and C alpha mRNAs were mainly expressed in primary spermatocytes. A postmeiotic expression predominating in early spermatids was unique to RII alpha mRNA. The location of RI, RII alpha, and C subunits in well-defined organelles of ram spermatids and epididymal sperm was assessed by immunogold electron microscopy. In spermatids, RI, RII alpha, and C were essentially present in the forming acrosome and, to a lesser extent, in the nucleus. During sperm epididymal maturation, the protein kinases disappeared from the acrosome and were detected in a variety of sperm functional areas, such as the tip of the acrosome, the motility apparatus, and the membrane network. The present study on subunits of cAMP-dependent protein kinase supports the concept that specific functions are attached to the different subunits in that it shows differential expression and differential subcellular localization in germ cells.
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PMID:Differential expression and subcellular localization for subunits of cAMP-dependent protein kinase during ram spermatogenesis. 276 39

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

We have shown previously that the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is an integral component of the mammalian sperm flagellum (Horowitz, J.A., H. Toeg, and G.A. Orr. 1984. J. Biol. Chem. 259:832-838; Horowitz, J.A., W. Wasco, M. Leiser, and G.A. Orr. 1988. J. Biol. Chem. 263:2098-2104). The subcellular localization of this flagellum-associated RII in bovine caudal epididymal sperm was analyzed at electron microscope resolution with gold-conjugated secondary antibody labeling techniques using anti-RII monoclonal antibodies. By immunoblot analysis, the flagellum-associated RII was shown to interact with mAb 622 which cross reacts with both neural and nonneural isoforms of RII. In contrast, a neural specific monoclonal antibody (mAb 526) failed to interact with flagellar RII. In the midpiece of the demembranated sperm tail, gold label after mAb 622 incubation was primarily associated with the outer mitochondrial membrane. Although almost all specific labeling in the midpiece can be assigned to the mitochondria, in the principal piece, there is some labeling of the fibrous sheath. Labeling of the outer dense fibers and the axoneme was sparse. Specific labeling was virtually absent in the sperm head. Sections of sperm tails incubated in the absence of primary antisera or with mAb 526 showed little labeling. A beta-tubulin monoclonal antibody localized only to the 9 + 2 axoneme. These results raise the possibility that a type II cAMP-dependent protein kinase located at the outer mitochondrial membrane plays a role in the direct cAMP stimulation of mitochondrial respiration during sperm activation.
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PMID:Immunogold localization of the regulatory subunit of a type II cAMP-dependent protein kinase tightly associated with mammalian sperm flagella. 297 31

The photoaffinity analog [32P]8-N3 cAMP (8-azido adenosine 3',5'-monophosphate was used to analyze the membrane sidedness of rat sperm cAMP binding proteins during epididymal maturation. Evidence is presented here which supports the hypothesis that 35-45% of the regulatory subunits of the Type I and Type II cAMP-dependent protein kinases are readily available to externally added cyclic nucleotide. It was observed by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and autoradiography that only two rat sperm proteins (Mr = 49K and 55K) were photolabeled which comigrated on gels with partially purified Type I and Type II regulatory subunits, respectively. Both of these photolabeled epididymal sperm proteins were saturated at physiological titers of [32P]8-N3cAMP and photoincorporation of [32P]8-N3 cAMP was specific since other SDS-resolvable sperm proteins did not photoincorporate the analog. Caput and cauda sperm protein photoincorporation could be effectively blocked by low levels of cAMP, but not by cGMP, ATP or GTP. Sperm epididymal maturation coincided with changes in the cAMP-dependent protein kinase subunits since cauda sperm contained more available Type II than did caput sperm. A subcellular analysis of cAMP-dependent protein kinase regulatory subunit in head and tail fractions was done for caput and cauda sperm and demonstrated that the tail fractions showed more photo-labeling of both Type I and II regulatory subunits than did the head fractions.
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PMID:A study of rat epididymal sperm adenosine 3',5'-monophosphate-dependent protein kinases: maturation differences and cellular location. 298 32


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