Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P56851 (epididymal)
 11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The reversible deactivation of chicken adipose tissue hormone-sensitive lipase alpha(previously activated with Mg2+ ATP and adenosine 3':5'-monophosphate) required Mg2+ and was inhibited by phosphate. These results are consistent with the assumption that deactivation of the protein kinase-activated enzyme is catalyzed by a lipase phosphatase. Cholesterol ester is catalyzed by a lipase phosphatase. Cholesterol ester hydrolase similarly was activated and reversibly deactivated. The activity of endogenous lipase phosphatase in pH 5.2 precipitate fractions was reduced, and in some cases eliminated, by incubation at 50 degrees for 20 min in buffer containing 20% glycerol. Heating at 50 degrees greatly increased the apparent percentage activation of triglyceride and cholesterol ester hydrolases but this was due to a selective decrease in basal (nonactivated) hydrolase activities. Essentially all endogenous lipase phosphatase could be removed by treatment of the pH 5.2 precipitate fraction with ATP-Sepharose affinity gel. The addition of a partially purified preparation of rat liver phosphorylase phosphatase deactivated triglyceride and cholesterol ester hydrolases. The deactivation process was concentration, 5 mM) and was inhibited by 5 mM phosphate and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipase alpha was also observed with crude prepa- and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipas alpha was also observed with crude preparations of phosphoprotein phosphatases from rat and turkey hearts, and from rat epididymal fat pads. Thus, hormone-sensitive lipase is deactivated by a variety of phosphoprotein phosphatases from different tissues and different species, implying a low degree of specificity for the deactivating system.
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PMID:Role of phosphoprotein phosphatases in reversible deactivation of chicken adipose tissue hormone-sensitive lipase. 19 Feb 35

1. Earlier studies have shown that exposure of fat-cells to insulin results in the rapid increased phosphorylation of an acid-soluble 22 kDa protein and that increases in phosphorylation were also evident in cells exposed to adrenaline [Belsham & Denton (1980) Biochem. Soc. Trans. 8, 382-383; Belsham, Brownsey, Hughes & Denton (1980) Diabetologia 18, 307-312]. 2. The effects of adrenaline are shown to be brought about through beta-adrenergic receptors and to be mimicked by other agents which increase cell cyclic AMP concentrations. The maximum extent of phosphorylation is about 60% of that observed with insulin. Increased phosphorylation is also observed in fat-cells exposed to vasopressin, oxytocin and phorbol esters, but not to alpha-adrenergic agonists. 3. No changes in the phosphorylation of the protein are evident in epididymal fat-pads from fat-fed, starved or starved/refed animals, despite the large changes in protein composition of fat-cells which accompany these nutritional alterations. This suggests that the protein is not closely involved in lipogenesis or associated metabolic pathways, but rather that it may play a more general regulatory role. 4. The 22 kDa protein migrates as a doublet on SDS/PAGE even after purification to apparent homogeneity by sequential use of Mono Q chromatography, SDS/PAGE and h.p.l.c. The amino acid compositions of the two components are very similar and share features in common with a number of proteins, including inhibitor-1, inhibitor-2, dopamine- and cyclic-AMP-regulated phosphoprotein (DARPP-32), and G-substrate, which may be involved in the regulation of protein phosphatase activity. 5. Phosphopeptide mapping and phosphoamino acid analysis reveals that insulin increases the phosphorylation of two distinct peptides within the protein (in one peptide insulin increases the amount of phosphothreonine, whereas in the other the hormone increases the amounts of phosphothreonine and phosphoserine). Both components of the doublet exhibit similar changes in phosphorylation, and hence the differences in migration are not the result of differences in phosphorylation, as suggested previously [Blackshear, Nemenoff & Avruch (1983) Biochem. J. 214, 11-19]. The pattern of phosphorylation observed with the beta-adrenergic agonist isoprenaline was similar to that observed with insulin. 6. The possible role and regulation of the 22 kDa protein are discussed.
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PMID:Comparison of the effects of insulin and adrenergic agonists on the phosphorylation of an acid-soluble 22 kDa protein in rat epididymal fat-pads and isolated fat-cells. 134 72

We have identified a bovine sperm phosphoprotein, pp255 (Mr = 255,000), which reacts strongly and specifically with an antibody to rat brain microtubule-associated protein 2 (MAP2). The phosphorylation state of this putative sperm MAP2 in intact bovine epididymal sperm is uniquely sensitive to regulation by intracellular pH (pHi), calcium, isobutyl-3-methylxanthine (MIX), H-8, and fluoride. Increasing pHi by approximately 0.4 units or exposure to calcium (0.1 microM with the ionophore A23187) or to the protein kinase inhibitor, H-8, decreases sperm MAP2 phosphorylation. Decreasing sperm pHi or exposure to MIX or fluoride increases MAP2 phosphorylation. Numerous other detectable sperm phosphoproteins are either unresponsive to most of these modulators or are considerably less sensitive to them. This phosphoprotein co-sediments with the particulate sperm heads during subcellular fractionation, and is not detectable in other sperm fractions. Two-dimensional electrophoresis separates sperm MAP2 into multiple species, indicative of varying degrees of phosphorylation. Sperm MAP2 is phosphorylated on serine residues, changes electrophoretic mobility slightly on one-dimensional gels with changes in phosphorylation levels, and exhibits the highest specific radioactivity of any sperm phosphoprotein observed. The phosphorylation state of sperm MAP2 can be uncoupled from sperm motility levels under several conditions. The co-localization of sperm MAP2 with the head fraction and the unique sensitivity of its phosphorylation level to modulators, which are known to regulate capacitation and the acrosome reaction, suggest that sperm MAP2 phosphorylation may be an intermediate step in the regulation of one or both of these sperm processes.
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PMID:The phosphorylation of a putative sperm microtubule-associated protein 2 (MAP2) is uniquely sensitive to regulation. 170 44

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

Bovine sperm in neat caudal epididymal fluid become motile in response to either pH elevation or dilution of the fluid. Buffers containing permeant weak acids at physiologic concentrations are able to mimic these effects of caudal fluid. These observations lead to the hypothesis that a pH-dependent epididymal fluid quiescence factor regulates bovine sperm motility by modulating sperm intracellular pH (pHi). Here we report that sperm pHi, measured with the fluorescent pH probe carboxyfluorescein, increases by approximately 0.4 units in response to either of these motility-initiating manipulations. At least 26 discrete phosphoprotein bands are distinguishable by sodium dodecylsulfate-polyacrylamide gel electrophoresis after incubation of intact caudal sperm with 32PO4. A prominent phosphoprotein, with Mr approximately 255,000 (pp255) and a relatively high specific radioactivity, is reversibly dephosphorylated in response to elevations in pHi that initiate sperm motility. Unlike most of the sperm phosphoproteins, the extraction of pp255 requires reducing agents. This phosphoprotein cosediments with the sperm heads but not the tail, midpiece, soluble, or plasma membrane fractions. No other pHi-dependent phosphorylation changes are apparent in gels of whole sperm extracts. However, subcellular fractionation allows the detection of increased phosphorylation of two plasma membrane phosphoproteins (Mr approximately 105,000 and 97,000) and decreased phosphorylation of another plasma membrane phosphoprotein (Mr approximately 120,000) in response to increasing pHi. This is the first report describing changes in endogenous phosphoproteins from intact motile and nonmotile bovine sperm that are regulated by pHi.
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PMID:Intracellular pH regulates bovine sperm motility and protein phosphorylation. 262 55

The fibrous sheath from rat epididymal sperm was isolated by sequential extraction, first with Triton X-100 and dithiothreitol, and then with 6 M urea and dithiothreitol. The latter extraction procedure solubilized most of the sperm components, leaving the head and the fibrous sheath as the only intact structures. This material was purified by sucrose gradient centrifugation. Electron microscopy confirmed the purity of the isolated material and revealed the characteristic structural features of the fibrous sheath. Polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulfate) of the fibrillar material, showed a complex polypeptide composition. The polypeptides with molecular weights of 80,000, 24,000, and 11,500 accounted for about 65% of the total protein of the fibrous sheath. Peptide map analyses indicated that the components of molecular weights of 80,000 and 24,000 are unrelated to the polypeptides of similar size of the outer dense fibers. On the other hand, it appears that the fibrous sheath and the outer dense fibers share the polypeptide of 11,500 daltons. The component of 80,000 daltons contains on the average about 3 mol of phosphoserine per mol of polypeptide, indicating that the most abundant polypeptide of the fibrous sheath is a phosphoprotein.
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PMID:The major component of the rat sperm fibrous sheath is a phosphoprotein. 270 27

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

An obstacle to the study of protein phosphorylation in mammalian spermatozoa has been the inability to incorporate sufficient amounts of 32Pi into cellular adenosine triphosphate (ATP) (Babcock et al., 1975). We report conditions under which 32Pi is effectively incorporated into the ATP of intact bovine spermatozoa. In the presence of a bicarbonate-buffered medium containing glucose, spermatozoa incorporated 32P into intracellular ATP in a time-dependent manner; after 2 h of incubation, the specific activity of [gamma-32P]ATP (2.3 X 10(4) cpm/nmol ATP) was estimated to be 50-65% of the specific activity of the intracellular phosphate pool. In the absence of glucose or other added substrates, the specific activity of [gamma-32P]ATP was 10-25% that of the specific activity observed in the presence of glucose. Washed spermatozoa incubated in carrier-free 32Pi for 2 h at 37 degrees C, and solubilized in a solution containing final concentrations of 6.8 M urea, 6% NP4O, and 5% beta-mercaptoethanol contained in excess of 40 32Pi-labeled proteins as assessed by two-dimensional polyacrylamide gel electrophoresis. Major phosphoproteins had approximate molecular weights of 93,000, 40,000, and 22,000. A different two-dimensional gel pattern was observed when cells were extracted with a solution containing 38.5 mM 2[N-cyclohexylamino] ethanesulfonic acid (CHES), pH 9.5/1.5% sodium dodecyl sulphate (SDS) at 100 degrees C. In contrast to the urea/Nonidet P-40 (NP40)/beta-mercaptoethanol extract, a 56,000 Mr phosphoprotein represented a major component while the 40,000 Mr and several of the 22,000 Mr polypeptides were markedly reduced in radioactive intensity. The 56,000 Mr species present in the CHES/SDS extract comigrated with the purified, phosphorylated regulatory subunit (RII) of cyclic adenosine 3',5'-monophosphate-dependent protein kinase from bovine heart. Antibodies to RII immunoprecipitated a 56,000 Mr, 32P-labeled polypeptide from the CHES/SDS extract that comigrated with purified, [32P] RII after two-dimensional electrophoresis. RII, then, appears to represent one of the endogenous phosphoproteins of intact bovine epididymal spermatozoa.
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PMID:Protein phosphorylation in intact bovine epididymal spermatozoa: identification of the type II regulatory subunit of cyclic adenosine 3',5'-monophosphate-dependent protein kinase as an endogenous phosphoprotein. 365 44

Isolated adipocytes from rat epididymal fat-pads were incubated with [32P]Pi, and intracellular phosphoproteins were then analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. A phosphorylated polypeptide of apparent Mr 46,000 was identified as the alpha-subunit of branched-chain 2-oxo acid dehydrogenase complex by immunoprecipitation using antiserum raised against the homogeneous E1 component of branched-chain 2-oxo acid dehydrogenase complex. Immunoprecipitation of this phosphoprotein is blocked in a competitive manner by purified branched-chain 2-oxo acid dehydrogenase complex. Peptide mapping of the isolated phosphoprotein indicates that two sites on the polypeptide are phosphorylated in the intact cells. Addition of branched-chain 2-oxo acids to the incubation medium causes diminution in the extent of labelling of both phosphorylation sites on the alpha-subunit, an effect presumably mediated via their known inhibitory action on branched-chain 2-oxo acid dehydrogenase kinase. These observations provide direct evidence for phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in intact cells.
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PMID:Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated adipocytes. Effects of 2-oxo acids. 379 71

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


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