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)

We have investigated the effects of adenosine on protein phosphorylation in extracts of rat heart. Incubation of a myofibrillar fraction with [gamma-32P]ATP resulted in the phosphorylation of several proteins by endogenous protein kinases. The adenosine analog 5'-chloro-5'-deoxyadenosine inhibited the phosphorylation of a 29 kD protein in this preparation. The protein was identified as cardiac troponin I (cTnI) by two-dimensional gel electrophoresis, using purified cTnI as standard. Addition of the catalytic subunit of cAMP-dependent protein kinase to the myofibrillar fraction increased phosphorylation of cTnI; this increase was inhibited by 5'-chloro-5'-deoxyadenosine and adenosine. Phosphorylation of purified cTnI by the catalytic subunit was also inhibited by 5'-chloro-5'-deoxyadenosine. Under these conditions used, 50% inhibition of phosphorylation by either endogenous or exogenous kinase was observed at approximately 50 microM 5'-chloro-5'-deoxyadenosine or adenosine. The inhibition described here occurred independently of catecholamines. The effects of ADP, AMP, and adenine on cTnI phosphorylation are also described.
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PMID:Inhibition of phosphorylation of troponin I in rat heart by adenosine and 5'-chloro-5'-deoxyadenosine. 185 69

Pretreatment of partially purified inhibitory GTP-binding protein (Gi, 41 kDa) with activated cyclic AMP-dependent protein kinase (PKA) decreases its ADP-ribosylation by islet-activating protein (pertussis toxin, IAP). We examined whether this decrease was associated with dissociation of the trimer of alpha beta gamma-subunits of Gi protein into alpha-subunits and beta gamma-subunits. Results showed that phosphorylation of the Gi protein by PKA impaired its dissociation into alpha-subunits and beta gamma-subunits by 50 mM Mg2+ and 100 microM GTP gamma S. The results suggested that phosphorylation of the Gi protein by PKA possibly caused a conformational change of the trimer Gi protein.
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PMID:Phosphorylation of Gi protein by cyclic AMP-dependent protein kinase inhibits its dissociation into alpha-subunits and beta gamma-subunits by Mg2+ and GTP gamma S. 190 64

Male Sprague-Dawley rats administered with an acute sublethal dose of carbofuran (1.5 mg/kg, s.c.) developed the signs of peak hypercholinergic activity during 30-60 min. At this time, in hemidiaphragm muscle, a significant decrease in ATP (28%) and phosphocreatine (PC) (29%) occurred without concurrent change in AMP and creatine (CR). A significant decrease in the levels of total adenine nucleotides (ATP + ADP + AMP) (20%) and total creatine compounds (PC + CR) (17%) was evident. The decline in the corresponding ratios of ATP/ADP (26%), ATP/AMP (39%), and PC/CR (20%) was therefore suggestive of greater utilization of ATP and PC in response to their increased demand for high-frequency muscle fasciculations. The energy charge = ATP + 1/2 ADP/(ATP + ADP + AMP), an index of high-energy phosphate adequacy in hemidiaphragm, remained unchanged. A significant (p less than 0.01) increase in serum magnesium with no concurrent change in calcium was also evident. The observed higher activity (152%) of total CK (EC 2.7.3.2) in the serum induced by carbofuran was possibly a reflection of more than a twofold increase in CK-BB isoenzyme (CK-1) and 141% increase in CK-MM isoenzyme (CK-3), which also strengthens our findings of enhanced synthesis of ATP and PC. Increased levels of CK-MM isoenzyme in the brain (253%) and hemidiaphragm (195%); and depletion of CK-BB isoenzyme in the hemidiaphragm (0%), heart (42%), and brain (77%), and of CK-MB isoenzyme (CK-2) in the brain (4%) and hemidiaphragm (14%), appeared to be the major contributory factors leading to enhanced serum CK activity.
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PMID:Carbofuran-induced alterations (in vivo) in high-energy phosphates, creatine kinase (CK) and CK isoenzymes. 195 49

Hepatocytes contain the Gi2 and Gi3 forms of the 'Gi-family' of guanine-nucleotide-binding proteins (G-proteins), but not Gi1. The anti-peptide antisera AS7 and I3B were shown to immunoprecipitate Gi2 and Gi3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein Gs. Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin-II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the alpha-subunit of immunoprecipitated Gi2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity ('Gi'-function). The immunoprecipitation of phosphorylated Gi-2 alpha-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of Gz (a 'Gi-like' G-protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the Gi family), in the immunoprecipitation assay. No labelling of the alpha-subunits of either Gi3 or Gs was observed. alpha-Gi2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of alpha-Gi2 was markedly biphasic where the loss of Gi function paralleled the high-affinity component of the labelling of alpha-Gi2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of alpha-Gi2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 microM) or 8-bromocyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated alpha-Gi2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [gamma-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated alpha-Gi2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of alpha-Gi2 in intact cells. We suggest that there are two possible sites for the phosphorylation of alpha-Gi2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.
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PMID:Hormonal regulation of Gi2 alpha-subunit phosphorylation in intact hepatocytes. 211 93

We studied the effect of adenosine nucleotides on several aspects of the functional activation of human peripheral blood polymorphonuclear leukocytes (PMN). Radiolabeled ATP bound to PMN in a manner suggesting the existence of specific binding sites because: 1) binding was reversed (92 +/- 6%) by 100-fold excess concentrations of unlabeled ATP but minimally by either ADP (43 +/- 12%) or GTP (37 +/- 8%); and 2) binding saturation was achieved (i.e., specific binding did not increase) above 250 microM ATP. Binding studies revealed that significant ATP hydrolysis occurred, even at low temperatures and in the presence of phosphatase inhibitors. Adenosine nucleotides activated signal transduction mechanisms in PMN because: 1) 1 to 100 microM ATP and 5'-adenylylimidodiphosphate (AMP-PNP) stimulated increased production of 1,2-diacylglycerols; 2) ATP (0.5 to 500 microM) and ADP (0.1 to 10 mM) induced increased insoluble protein kinase (PKC) activity in a dose-dependent manner when used at concentrations greater than 50 microM; 3) ATP (greater than or equal to 50 microM) induced a shift in the solubility of phorbol receptors from mostly soluble (89% in untreated cells) to mostly insoluble (68%), whereas ADP, GTP, and GDP were effective at higher concentrations; and 4) greater than or equal to 50 microM ATP stimulated increased phosphorylation of endogenous PMN proteins. AMP-PNP induced PKC activity and phosphoprotein changes that were qualitatively similar to those observed when PMN were treated with ATP, suggesting that extracellular ATP hydrolysis is not required for signal transduction to activate PKC. Functionally, ATP stimulated the secretion of specific (but not azurophil) granules because vitamin B12-binding protein and low levels of lysozyme, but not beta-glucuronidase, were released; qualitatively similar results were obtained by using AMP-PNP. These results suggest that certain adenosine nucleotides employed at physiologically relevant concentrations stimulate increased 1,2-diacylglycerol production, PKC activity, granule secretion, and endogenous phosphoprotein formation in a manner that is independent of extracellular ATP hydrolysis.
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PMID:Extracellular adenosine nucleotides stimulate protein kinase C activity and human neutrophil activation. 215 72

Effects of ATP on junctional conductance (gj) were investigated in paired ventricular myocytes isolated from guinea pig hearts. One cell of the pair was voltage-clamped with a single-patch pipette, and gj was measured after the perforation of the nonjunctional membrane of the partner cell. The current-voltage relation of gj was linear between -30 and +30 mV. The control gj at 5.0 mM ATP in 88 pairs of cells ranged from 100 to 1,055 nS (average, 268 nS). ATP within the range from 0.1 to 5.0 mM increased gj in a dose-dependent manner. The Hill coefficient was 2.6, and the half-maximum effective concentration of ATP was 0.68 mM. Adenylylimidodiphosphate (2 mM) caused a transient increase in gj in the presence of 0.5 mM ATP, but forskolin (30 microM), cyclic AMP (50 microM), catalytic subunit of cyclic AMP-dependent protein kinase (1 microM), and ADP (10 mM) had no significant effect on gj. The temperature coefficient of gj in the presence of 5.0 mM ATP was 1.29. These findings suggest that gj in paired ventricular myocytes is directly regulated by ATP probably through a specific ligand-receptor interaction between ATP and gap junctional channel protein.
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PMID:ATP directly affects junctional conductance between paired ventricular myocytes isolated from guinea pig heart. 215 33

The kinetic reaction mechanism of the type II calmodulin-dependent protein kinase was studied by using its constitutively active kinase domain. Lacking regulatory features, the catalytic domain simplified data collection, analysis, and interpretation. To further facilitate this study, a synthetic peptide was used as the kinase substrate. Initial velocity measurements of the forward reaction were consistent with a sequential mechanism. The patterns of product and dead-end inhibition studies best fit an ordered Bi Bi kinetic mechanism with ATP binding first to the enzyme, followed by binding of the peptide substrate. Initial-rate patterns of the reverse reaction of the kinase suggested a rapid-equilibrium mechanism with obligatory ordered binding of ADP prior to the phosphopeptide substrate; however, this apparent rapid-equilibrium ordered mechanism was contrary to the observed inhibition by the phosphopeptide which is not supposed to bind to the kinase in the absence of ADP. Inspection of product inhibition patterns of the phosphopeptide with both ATP and peptide revealed that an ordered Bi Bi mechanism can show initial-rate patterns of a rapid-equilibrium ordered system when a Michaelis constant for phosphopeptide, Kip, is large relative to the concentration of phosphopeptide used. Thus, the results of this study show an ordered Bi Bi mechanism with nucleotide binding first in both directions of the kinase reaction. All the kinetic constants in the forward and reverse directions and the Keq of the kinase reaction are reported herein. To provide theoretical bases and diagnostic aid for mechanisms that can give rise to typical rapid-equilibrium ordered kinetic patterns, a discussion on various sequential cases is presented in the Appendix.
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PMID:Kinetic mechanism of the type II calmodulin-dependent protein kinase: studies of the forward and reverse reactions and observation of apparent rapid-equilibrium ordered binding. 215 78

SQ-27986, a oxabicycloheptane derivative, potently inhibits ADP-, collagen- and arachidonic acid-induced platelet aggregation in human platelet-rich plasma. Human platelet aggregation induced by ADP is inhibited by SQ-27986 (EC50 = 22nM), and the inhibitory action of SQ-27986 can be prevented with N-0164, a PGD2 antagonist. By comparison, ADP-induced rat platelet aggregation is unaffected by SQ-27986 (IC50 greater than 80 microM). Washed human platelets treated with SQ-27986 exhibit elevated cAMP levels and activated cAMP-dependent protein kinase. Elevation of platelet cAMP levels (greater than 4 fold basal) and activation of the cAMP-dependent protein kinase (greater than 4 fold) are observed with SQ-27986 concentrations above 100 nM. The SQ-27986-induced elevation of cAMP can be prevented by N-0164. Lysed platelets treated with SQ-27986 showed stimulated adenylate cyclase activity. SQ-27986 competes with [3H]prostaglandin D2 binding to isolated platelet membranes (EC50 for SQ-27986 is 20 nM, which was more potent than cold PGD2 itself). Radiolabeled Iloprost binding is virtually unaffected by SQ-27986 (EC50 greater than 100 microM), indicating that SQ-27986 does not interact with platelet prostacyclin receptors. These studies indicate that SQ-27986 inhibits platelet aggregation by activating platelet adenylate cyclase via stimulation of platelet PGD2 receptors.
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PMID:SQ-27986 inhibition of platelet aggregation is mediated through activation of platelet prostaglandin D2 receptors. 217 Oct 39

Octimibate inhibited ADP- and collagen-induced platelet aggregation in human, rabbit and rat platelet-rich plasma. Washed human platelets treated with octimibate had elevated cyclic AMP (cAMP) levels and cAMP-dependent protein kinase activity. When whole platelets were incubated with radiolabeled phosphate, octimibate produced an increase in the phosphorylation of platelet proteins with relative molecular weights of 22, 26, 50 and 80 kilodaltons. This pattern of protein phosphorylation is identical to that observed when the platelets were treated with forskolin, phosphodiesterase inhibitors or other compounds that elevate platelet cAMP levels. Octimibate also inhibited the rise in intracellular Ca++ caused by thrombin, as measured using Fura-2-loaded platelets, which is consistent with octimibate's ability to elevate platelet cAMP levels. When isolated platelet plasma membranes were treated with octimibate, adenylate cyclase activity was stimulated, reaching maximal activation at 1 microM octimibate. (The maximal activation of adenylate cyclase observed with octimibate is 70-75% of that observed with 10 microM PGE1.) This stimulation of platelet adenylate cyclase activity was enhanced by GTP. Octimibate competed for radiolabeled prostaglandin E1 and lloprost binding to isolated platelet membranes at submicromolar concentrations, but did not compete with radiolabeled prostaglandin D2 binding. These studies suggest that octimibate inhibits platelet aggregation by activating platelet adenylate cyclase through stimulation of platelet prostacyclin receptors.
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PMID:Octimibate inhibition of platelet aggregation: stimulation of adenylate cyclase through prostacyclin receptor activation. 217 92

Quantitative and qualitative changes in adrenoceptors under various conditions were studied by binding experiments. Chronic treatment with reserpine increased the level of alpha 2-adrenoceptors in rat vas deferens and hypoxia increased the level of alpha 1-adrenoceptors in rat cardiomyocytes. Adenosine receptor agonists increased the affinity of the alpha 2-adrenoceptor in rat vas deferens for the agonist with an increase in receptor-mediated responses. Thus two types of changes in receptor binding sites were observed. Next, changes in the GTP-binding (G) protein were studied. Activation of cyclic AMP-dependent protein kinase (PKA) decreased the ADP-ribosylation of Gi (41 K) protein by islet-activating protein (pertussis toxin, IAP). Purified Gi protein was phosphorylated by the enzyme. IAP-sensitive G protein-mediated coupling responses such as phosphatidylinositol turnover in differentiated HL-60 cells were also modified under this condition. These results indicated that phosphorylation of Gi by PKA caused a qualitative change of Gi. Lithium ions also decreased the ADP-ribosylation of Gi by IAP. Then it determined if the decrease was accompanied with a dissociation of the subunits of Gi. Phosphorylation of Gi by PKA impaired the dissociation of the subunits of Gi caused by Mg2+ and GTP gamma S, whereas lithium ions did not have any effect on their dissociation. Thus some conditions caused a functional change in the so-called "qualitative change" of Gi.
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PMID:The mechanism of changes in adrenoceptor-mediated responses. 217 4


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