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)

Incubation of a hepatocyte particulate fraction with ATP and the isolated catalytic unit of cyclic AMP-dependent protein kinase (A-kinase) selectively activated the high-affinity 'dense-vesicle' cycle AMP phosphodiesterase. Such activation only occurred if the membranes had been pre-treated with Mg2+. Mg2+ pre-treatment appeared to function by stimulating endogenous phosphatases and did not affect phosphodiesterase activity. Using the antiserum DV4, which specifically immunoprecipitated the 51 and 57 kDa components of the 'dense-vesicle' phosphodiesterase from a detergent-solubilized membrane extract, we isolated a 32P-labelled phosphoprotein from 32P-labelled hepatocytes. MgCl2 treatment of such labelled membranes removed 32P from the immunoprecipitated protein. Incubation of the Mg2+-pre-treated membranes with [32P]ATP and A-kinase led to the time-dependent incorporation of label into the 'dense-vesicle' phosphodiesterase, as detected by specific immunoprecipitation with the antiserum DV4. The time-dependences of phosphodiesterase activation and incorporation of label were similar. It is suggested (i) that phosphorylation of the 'dense-vesicle' phosphodiesterase by A-kinase leads to its activation, and that such a process accounts for the ability of glucagon and other hormones, which increase intracellular cyclic AMP concentrations, to activate this enzyme, and (ii) that an as yet unidentified kinase can phosphorylate this enzyme without causing any significant change in enzyme activity but which prevents activation and phosphorylation of the phosphodiesterase by A-kinase.
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PMID:Activation and phosphorylation of the 'dense-vesicle' high-affinity cyclic AMP phosphodiesterase by cyclic AMP-dependent protein kinase. 254 54

We show that promastigotes of Leishmania donovani, the causative agent of visceral leishmaniasis (kala-azar), possess heparin receptors on their surface. From a linear Scatchard plot of the binding data obtained using [3H]heparin and viable promastigotes, one derives a binding constant of 4.7 x 10(-7) M and an estimate of 860,000 receptors per parasite. The [3H]heparin bound to parasites could not be displaced by hyaluronic acid or by three other glycosaminoglycans (dermatan sulphate, chondroitin 4-sulphate and chondroitin 6-sulphate). It was demonstrated that exponential phase promastigotes growing in medium 199 supplemented with fetal bovine serum incorporate 35SO4 into a cell-associated macromolecule that has the properties of heparin proteoglycan. Heparin inhibits the activity of the cell-surface histone-protein kinase; incubation of viable promastigotes with [gamma-32P]ATP and MgCl2 (10 mM) in the absence and presence of heparin (0.01-0.5 mg/ml) for 10 min, followed by analysis by SDS/polyacrylamide-gel electrophoresis and autoradiography, revealed that the phosphorylation of 12 or 13 parasite proteins was inhibited by the glycosaminoglycan. These data suggest that heparin may play a role in the host-parasite relationship.
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PMID:Heparin binds to Leishmania donovani promastigotes and inhibits protein phosphorylation. 255 42

The molecular basis of opioid receptor mechanisms was studied in reconstitution experiments using purified or membrane-bound opioid receptors and purified GTP-binding proteins (G-proteins). mu-Opioid receptor exclusively purified from rat brains was reconstituted with G-proteins in lipid vesicles. The mu-agonist stimulated the G-protein activity in both G1 or Go-reconstituted vesicles. The stoichiometry revealed that one molecule of mu-receptor is functionally coupled to plural numbers of Gi or Go molecules and that mu-receptor exists in at least two different subtypes, mu i and mu o, separately coupled to Gi and Go, respectively. In addition, when the mu-receptor was phosphorylated by cAMP-dependent protein kinase, the mu-agonist-stimulation of G-protein activity disappeared, while the guanine nucleotide-sensitivity of agonist binding was unchanged. These findings suggest that there are independent domains in the receptor which are related to functional coupling to G-protein and to the agonist-binding modulation by G-protein. kappa-Opioid receptor agonist inhibited the G-protein activity in guinea pig cerebellar membranes. Further experiments revealed that the kappa-opioid receptor is functionally coupled to an inhibition of phospholipase C activity via an inhibition of Gi-activity. Such a receptor-mediated inhibition of G-protein activity may be the first demonstration of a signal transduction mechanism. The delta-opioid receptor agonist showed no effect on G-protein activity in guinea pig striatal and rat cortical membranes, while it stimulated it in NG108-15 cells. In all these membranes, the delta-agonist binding was markedly reduced by GTP gamma S in the presence of MgCl2. These findings suggest that delta-receptors in the brain might be coupled to G-protein without signal transduction.
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PMID:[Molecular pharmacology of opioid receptor mechanisms]. 255 62

Protein kinase C (PKC) is a Ca2+- and phospholipid-dependent protein kinase which has been implicated as a key enzyme in the regulation of cellular growth. The naphthalenesulfonamide W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] is representative of a number of cationic amphiphilic inhibitors of PKC which appear to inhibit PKC by interacting with the acidic phospholipid cofactor of the enzyme, according to kinetic studies. In a previous report, we demonstrated that PKC binds directly to W7 when the naphthalenesulfonamide is immobilized on agarose. In the present report, we have defined the mechanism of the binding of PKC to W7-agarose, and its relevance to the inhibitory mechanism of the naphthalenesulfonamide. We demonstrate that PKC bound W7-agarose through the catalytic domain of the enzyme. An active catalytic fragment of PKC was generated by limited proteolysis, and we found that this fragment bound W7-agarose and coeluted with intact PKC upon the addition of Triton X-100. W7 inhibited PKC activity by two different mechanisms. As previously reported, W7 inhibited PKC by interacting with the phospholipid cofactor of the enzyme (IC50 = 260 microM). However, at higher concentrations of W7, we found that this naphthalenesulfonamide inhibited PKC by serving as a competitive inhibitor with respect to the substrate ATP, according to a kinetic analysis of the inhibition of the active catalytic fragment of PKC by W7. W7 inhibited the active catalytic fragment of PKC as well as PKC-catalyzed phosphorylation of protamine sulfate, a reaction which is independent of Ca2+ and phospholipid, with similar potencies. Consistent with the kinetic evidence that W7 serves as a competitive inhibitor of PKC with respect to ATP, we found that, in the presence of 10 mM MgCl2, 1 mM ATP was sufficient to elute PKC from W7-agarose. Thus, naphthalenesulfonamide PKC inhibitors may include both agents which primarily function by interacting with the phospholipid cofactor of the enzyme and agents which primarily serve as active site inhibitors of PKC.
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PMID:Binding of protein kinase C to N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide through its ATP binding site. 273 31

We have developed a one-step purification procedure for proteins containing the N-terminal portion of the gag protein of avian sarcoma and leukemia viruses. In this procedure, a resin with a covalently attached monoclonal antibody to the gag protein p19 is used to bind gag-containing proteins from crude extracts. After washing of the resin, the bound proteins are eluted with 2 M MgCl2. For the transforming protein kinase encoded by Fujinami sarcoma virus p130gag-fps, this procedure gave an enrichment of several thousand-fold, a yield of over 10%, a final purity of over 20%, and no significant loss of protein kinase activity. Similar purifications were obtained with three other gag-containing proteins. The immunoaffinity purification described may be of general utility as a first step in purification of the several other avian retroviral transforming proteins that are synthesized from fusions of an oncogene with the viral gag gene.
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PMID:A simple method for immunoaffinity purification of nondenatured avian sarcoma and leukemia virus gag-containing proteins. 282 89

Pretreatment of rat cardiac myocytes with the beta-adrenergic agonist, db-cAMP or forskolin decreased ADP-ribosylation of 40-41 kDa protein by islet-activating protein (IAP) in cell membranes. Addition of activated cyclic AMP-dependent protein kinase (protein kinase A) catalytic subunit and MgCl2 also decreased ADP-ribosylation of 40-41 kDa protein by IAP in cell membranes. The alpha- and beta-subunits of partially purified inhibitory GTP-binding protein (Gi) were both phosphorylated by protein kinase A. The amounts of phosphate incorporated into the subunits of Gi were 0.34 and 0.18 mol/mol protein. These show that phosphorylation of Gi by protein kinase A results in a decrease in its ADP-ribosylation by IAP.
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PMID:Effects of phosphorylation of inhibitory GTP-binding protein by cyclic AMP-dependent protein kinase on its ADP-ribosylation by pertussis toxin, islet-activating protein. 284 88

A study has been made of the action of the neuropeptide proctolin on the radiosodium efflux from single barnacle muscle fibers. Proctolin (10(-8) M) when applied externally causes stimulation of the Na efflux in unpoisoned and ouabain-poisoned fibers. The response is prompt in onset, reaches a peak in 15 min and decays slowly. The response of the ouabain-insensitive Na efflux to external proctolin is dose-dependent, the concentration threshold being less than 10(-10) M. The response to proctolin is dependent on external Ca2+ but not on Na+. (i) The response to proctolin is abolished by high external Mg2+, as well as by verapamil, Co2+, Cd2+ and WB-4101. (ii) The response is also abolished by preinjecting 0.5 M MgCl2 or 0.1 M EGTA. The calmodulin antagonists trifluoperazine and imipramine are without effect on the response to proctolin. (i) Adenylate cyclase agonists, e.g. forskolin, fail to augment the response to proctolin. (ii) Prior injection of the phosphodiesterase inhibitors 1-propyl-3-methyl-7-(5-hydroxyhexyl)-xanthine (PMX) or 1-isoamyl-3-isobutylxanthine (IAX) fails to augment the response to proctolin. (iii) Prior injection of protein kinase inhibitor is ineffective. The response to proctolin is significantly reduced in the presence of tyramine. Taken together, these results support the view that proctolin stimulates the ouabain-insensitive Na efflux by activating Ca2+ channels and that the cAMP-protein kinase system is not involved in this response.
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PMID:Stimulation by proctolin of the ouabain-insensitive sodium efflux in single barnacle muscle fibers. 286 54

Monoclonal antibodies have been raised against canine phospholamban purified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). Four of twenty-four antibodies were purified to close to homogeneity from mouse ascites. All four antibodies could react with isolated bovine cardiac sarcoplasmic reticulum (SR) to result in the stimulation of ATP-dependent Ca2+ pump activity and blocking of phospholamban phosphorylation by cAMP-dependent protein kinase. Relative efficiencies of antibodies in Ca2+ pump stimulation and on phospholamban phosphorylation were not correlated. An immunoabsorbent prepared by conjugating antibody Al to Affi-Gel 10 was used for the purification of phospholamban. Isolated bovine cardiac SR was solubilized in a buffer containing deoxycholate and the soluble fraction was applied to the immunoaffinity column. After washing the column with a series of detergent-containing buffer solutions, the column-bound protein which contained essentially pure phospholamban was eluted by a buffer containing 2.8 M MgCl2. The phospholamban recovery from the immunoaffinity column was close to 100%; the overall yield of purification from SR vesicles was about 70%. SDS-PAGE analysis showed that purified phospholamban consisted of a 25 and 5 kilodalton (kDa) protein species. Upon brief boiling (20 s) of the sample in SDS-PAGE sample buffer, five molecular species ranging from 5 to 25 kDa could be detected by immunotransblotting following SDS-PAGE. This observation supports the notion that phospholamban is composed of five 5-kDa polypeptides. The pure phospholamban could be phosphorylated maximally by cAMP-dependent protein kinase to 1-1.5 mol phosphate/mol phospholamban (25,000 g). This stoichiometry of phosphorylation could be increased to about 5 upon addition of the immunoaffinity column flow through fraction.
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PMID:Rapid purification of phospholamban by monoclonal antibody immunoaffinity chromatography. 295 97

Under the total blockade of PDE1 and the presence of endogeneous ATP and MgCl2, the inhibitory effect of cAMP on HD activity could be demonstrated as low as 8.7 X 10(-8) M concentration in a 20,000 g supernatant of a sustained homogenate of rat hypothalamus. A total reverse of this action and also a partial release of the cAMP-induced inhibition of HD, occurred at higher concentrations of cAMP, and ATP could be achieved by an endogeneous inhibitor of cAMP-dependent protein kinase or by cyclic GMP. The reversal of cAMP action by PKI seems to serve a strong evidence for the role of cAMP-dependent protein kinase (EC 2.7.37: ATP-protein phosphotransferase) in this action and emphasized the involvement of a direct or an indirect phosphorylation in the regulation of HD activity. The stimulatory effect of cyclic GMP on cAMP-induced inhibition of HD or its 'direct' effect on histamine formation is asserted, probably through the activation of PDE, or through independent stimulatory machinery, coupled to the cyclic GMP system.
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PMID:Evidence for the role of cAMP-dependent protein kinase in the down-regulation of hypothalamic HD: reversal of cAMP-(ATP) induced inhibition of HD activity by the 'Walsh' inhibitor of cAMP-dependent protein kinase and by cyclic GMP. 299 Jan 78

An extra-ribosomal cAMP-independent protein kinase from cryptobiotic embryos of Artemia salina has been purified to near homogeneity by gel filtration on Bio-Gel A-0.5 m, ion-exchange chromatography on DEAE-cellulose and phosphocellulose P11 and affinity chromatography on casein-Sepharose 4B and ATP-agarose. The enzymatic activity has a broad optimum at pH 7-8. Maximal activity is obtained in the presence of 5-6 mM MgCl2. The activity is inhibited by Mn2+, Ca2+ and K+. The enzyme has an Mr of 127 000, utilizes both ATP and GTP as phosphoryl donors and is completely inhibited by heparin and poly(L-glutamic acid). According to its properties, the enzyme can be classified as a casein kinase type II. Although the enzyme is associated with ribosomes, ribosomal proteins are not among the main substrates. The kinase is able to phosphorylate both the alpha and the beta subunits of initiation factor eIF2 using ATP or GTP as phosphoryl donors. The function of phosphorylation in the initiation of protein synthesis is discussed.
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PMID:Ribosome-associated cyclic nucleotide-independent protein kinase of Artemia salina cryptobiotic gastrulae. 299 May 57


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