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)

Purified inhibitor of the cyclic AMP-dependent protein kinase (PKI) has been used as a probe to determine if hormone and cyclic AMP-induced activation of the cardiac alkaline triacylglycerol (TG) lipase is mediated through the cAMP-dependent protein kinase. Addition of CAM (cyclic AMP, Mg-ATP, and 3-isobutyl, 1-methylxanthine) to any of the four fractions (homogenate, 10,000 g supernatant, 105,000 g supernatant, or heparin-Sepharose eluate) from heparin perfused heart activated the TG lipase 60% to 110%. Preincubation of these fractions with 33 ng of PKI had no effect on control enzyme activity. Addition of PKI (33 ng) to extracts following CAM activation had little effect on homogenate TG lipase activity, but reduced activities in 10,000 g and 105,000 g supernatant fractions to their respective control levels, and inhibited TG hydrolase activity of activated heparin-Sepharose eluate to 50% below the control activity. If extracts were preincubated with PKI prior to CAM addition, TG lipase activity was reduced to approximately 50% below control levels in all fractions. PKI addition (33 ng) to 105,000 g supernatant obtained from hearts stimulated 60% by epinephrine perfusion reduced activity to 50% below the control level. PKI inhibition of TG lipase activity of 105,000 g supernatant could be reversed by adding 0.5 microgram of catalytic subunit of protein kinase (PKC) to the extract. The inhibition below control levels caused by CAM and PKI indicate that the PKI-PKC complex by itself or in combination with other extract molecules, has an inhibitory effect on the TG lipase.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Cell Cardiol 1987 Jul
PMID:Protein kinase inhibitor blocks the activation of a myocardial triacylglycerol lipase. 282 94

The enzyme, myosin light chain kinase, has been purified to homogeneity from bovine aortic vascular smooth muscle. Approximately 10 mg of enzyme could be obtained from 1 kg of fresh aortas with an overall yield of 26% of the original activity. The vascular myosin light chain kinase has a molecular weight of 160 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Antiserum raised to the aortic myosin light chain kinase in rabbits strongly inhibited phosphotransferase activity. In addition, the antiserum was used to identify myosin kinase in a crude homogenate of vascular smooth muscle by radioimmunoblotting. A single species of the enzyme (Mr = 160 000) was identified. The bovine aortic myosin kinase could be phosphorylated by both cyclic AMP- and GMP-dependent protein kinases. Approximately 2 mols PO4/mole of enzyme could be incorporated by the cyclic AMP-dependent protein kinase in the absence of calmodulin. If Ca2+ and calmodulin were included in the reaction mixture, phosphate incorporation by the cyclic AMP-dependent protein kinase was reduced to 1 mol and phosphorylation by cyclic GMP-dependent protein kinase was completely inhibited. These results were confirmed by tryptic peptide mapping. Two distinct phosphopeptides were identified: site-1 and site-2. Both could be phosphorylated by the cyclic AMP-dependent protein kinase but only site-1 was phosphorylated by the cyclic GMP-dependent enzyme. In the presence of Ca2+ and calmodulin, phosphorylation by cAMP-dependent protein kinase was restricted to site-1. The effect of phosphorylation on myosin light chain kinase activity was determined. Only phosphorylation by cyclic AMP-dependent protein kinase was found to alter the requirement of myosin kinase for calmodulin. The K0.5 (i.e. the concentration of calmodulin required for half-maximal enzyme activation) for calmodulin was 5 nM for the unphosphorylated myosin kinase. With 2 mol PO4/mol myosin kinase incorporated, the K0.5 for calmodulin was increased to 82 nM. When only 1 mol PO4/mol myosin kinase was incorporated, no effect on calmodulin requirement was observed. Moreover, single site phosphorylation had no effect on other activity parameters, including Km for ATP and for light chains. Our studies suggest that cyclic AMP-dependent protein kinase may play an important role in the regulation of vascular myosin kinase activity. Moreover, our results indicate that cyclic GMP-dependent protein kinase does not affect calmodulin-activation of myosin kinase or several other activity parameters.
J Mol Cell Cardiol 1985 Sep
PMID:Phosphorylation of myosin light chain kinase from vascular smooth muscle by cAMP- and cGMP-dependent protein kinases. 299 88

A cardiac muscle sarcolemmal preparation, enriched in adenylate cyclase, Na+, K+ -ATPase, beta, muscarinic and ouabain receptors, also contained endogenous protein kinase activity. Phosphorylation of sarcolemmal membrane proteins by the endogenous protein kinase occurred mainly on 22 000 and 12 000 Mr proteins. To determine the effect of this phosphorylation on sarcolemmal properties, sarcolemmal vesicles were preincubated under conditions for optimal phosphorylation while control vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both control and phosphorylated vesicles were centrifuged, resuspended in 10 mM Tris-Cl (pH 7.4) and subsequently assayed for ATPase activities and for binding of ouabain, dihydroalprenolol and quinuclidinyl benzilate to the membranes. Sarcolemmal phosphorylation was associated with an increase in Ca2+ -ATPase activity but had no effect on Mg2+ ATPase or Na+, K+ -ATPase activity or on ouabain binding. Muscarinic receptor and beta-adrenoreceptor binding also appeared to be unaffected.
J Mol Cell Cardiol 1985 Nov
PMID:Influence of protein kinase phosphorylation on isolated sarcolemmal membranes. 300 20

In isolated rat hearts perfused at constant coronary flow and heart rate the amount of shortening in their major axis (L), the developed tension (T) and their time derivatives (L and T) were measured after different interventions. Relative changes of maximal velocities of contraction (+L, +T) and relaxation (-L, -T) were assessed by the ratio between both velocities (+L/-L, +T/-T). After the interventions, cAMP intracellular levels and cAMP dependent protein kinase activity were measured. The negative inotropic effect of verapamil (10(-7) M) and nifedipine (5 X 10(-7) M) was accompanied by a relatively greater decrease in maximal velocity of relaxation. Consequently the ratio +L/-L increased. Verapamil increased +L/-L from 1.27 +/- 0.07 to 1.57 +/- 0.08 (P less than 0.001). Nifedipine increased +L/-L from 1.25 +/- 0.05 to 1.77 +/- 0.12 (P less than 0.001). Whereas nifedipine increased intracellular cAMP levels from 0.403 +/- 0.036 pmol/mg wet weight to 0.534 +/- 0.047 (P less than 0.05), verapamil did not alter them. Neither verapamil nor nifedipine affected cAMP protein kinase activity. Increasing Ca2+ in the perfusate did not change the ratio +L/-L. A decrease in extracellular Ca2+, on the other hand, produced a greater decrease in -L than in +L, so that the ratio +L/-L increased from 1.27 +/- 0.05 to 1.49 +/- 0.10 (P less than 0.05). No changes were detected in cAMP levels or its protein kinase activity. Similar results were obtained when +T/-T was analyzed. To offset the negative inotropic effect caused by calcium antagonists, either increased extracellular Ca2+ or isoproterenol can be used.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Cell Cardiol 1985 Mar
PMID:The link between myocardial contraction and relaxation: the effects of calcium antagonists. 301 63

The various subtypes of adrenergic receptors represent distinct structural entities which are coupled in different ways to two major transmembrane signalling systems, the adenylate cyclase and phosphatidyl-inositol pathways. Recent evidence suggests that the functional linkage of both beta and alpha 1-adrenergic receptors to their respective effector systems is regulated by covalent modification of the receptors by phosphorylation-dephosphorylation reactions. Receptor phosphorylation appears to lead to desensitization of the biological response to receptor stimulation. Several kinases including protein kinase A, protein kinase C and a cAMP independent kinase appear to participate in these reactions.
J Mol Cell Cardiol 1986 Sep
PMID:Ciba-Geigy award for outstanding research. Regulation of adrenergic receptor function by phosphorylation. 302 44

The effect of cAMP-dependent protein kinase on calcium uptake and protein phosphorylation in bovine aortic microsomes was examined. Acid gel electrophoresis demonstrated that the aortic microsomes contained a Ca2+-dependent, hydroxylamine-sensitive phosphoenzyme (Mr 110 kDa), characteristic of the calcium pump in sarcoplasmic reticulum, but showed no evidence of a sarcolemmal calcium pump. Calcium uptake by these aortic vesicles was markedly stimulated by oxalate, whereas calcium uptake by canine cardiac sarcolemmal vesicles was oxalate-independent. Both cAMP plus protein kinase (cAMP-PK) and catalytic subunit of protein kinase stimulated oxalate-supported calcium uptake by bovine aortic microsomes 23 +/- 3% (P less than 0.05) at 0.3 microM Ca2+, but had no effect at 6 to 10 microM Ca2+. Catalytic subunit of protein kinase and cAMP-PK phosphorylated an 11 kDa protein in bovine aortic microsomes which comigrated with canine cardiac phospholamban after boiling in sodium dodecylsulfate. The stoichiometry of the aortic 11 kDa phosphoprotein to 110 kDa phosphoenzyme was approximately 1:1. These data are consistent with the recent identification of phospholamban in various smooth muscles, and suggest that cAMP-mediated vascular relaxation may in part be attributable to stimulation of calcium uptake by the sarcoplasmic reticulum.
J Mol Cell Cardiol 1988 Aug
PMID:Regulation of calcium uptake in bovine aortic sarcoplasmic reticulum by cyclic AMP-dependent protein kinase. 322 9

In the mammalian myocardium, an active triglyceride synthesis pathway is operating, (re)esterifying activated fatty acids from endogenous or exogenous sources, with the glycolytically derived three-carbon intermediates dihydroxyacetone-phosphate and glycerol-3-phosphate by the so-called Kennedy pathway. The seven enzymes of triglyceride synthesis are membrane bound and located at the sarcoplasmic reticulum. The first enzyme in the glycerol-3-phosphate pathway, glycerol-3-phosphate acyltransferase, is proposed to be rate limiting for triglyceride formation. This microsomal enzyme is regulated by phosphorylation (inactiycation)-dephosphorylation (activation) coupled to the beta-receptor--adenyl cyclase--protein kinase system. Additional regulatory steps in triglyceride formation are the reactions catalyzed by the microsomal phosphatidic acid phosphatase and diglyceride acyltransferase. Intracellular triglycerides occur as free floating cytosolic droplets, membrane-bound particles and lipid-filled lysosomes. No consensus exists about the metabolically active portion of myocardial triglycerides. Various lipases have been proposed to be involved in endogenous lipolysis: the lysosomal acid, microsomal and soluble neutral triglyceride, intracellular lipoprotein lipases and the microsomal di- and monoglyceridase. It has been acknowledged that the bulk of the intracellular neutral lipase represents the precursor of vascular lipoprotein lipase. The presence of a neutral lipase, as distinct from lipoprotein lipase, in the rat heart was recently advocated. Endogenous lipolysis is a hormone-sensitive process. Hormone-sensitivity may involve direct alteration of enzyme activity by protein phosphorylation-dephosphorylation but is also dependent on the removal rate of product fatty acids, since feedback inhibition is a common property of all lipases in the heart.(ABSTRACT TRUNCATED AT 250 WORDS)
Basic Res Cardiol 1987
PMID:Synthesis, storage and degradation of myocardial triglycerides. 331 Oct 5

Glycerol 3-phosphate acyltransferase (GPAT) activity and triglyceride lipase (TGL) activity were measured in homogenates from hearts perfused with adrenergic agonists and antagonists. Perfusion with adrenalin or the beta-agonist isoprenaline produced an increase in TGL activity and a fall in GPAT activity. These changes could be imitated by incubation of heart homogenates with cAMP-dependent protein kinase. The alpha 2-agonist clondine produced the opposite effect, thus it increased GPAT activity and decreased TGL activity. Methoxamine, an alpha 1-agonist, had no effect on TGL activity but reduced GPAT activity. Continuous perfusion of the beta-antagonist atenolol reduced TGL activity to half that found in controls but also reduced GPAT activity. No change was seen on continuous perfusion of alpha 1- or alpha 2-antagonists. Changes in GPAT activity were localized mainly in the microsomal enzyme. These changes are consistent with both enzymes being regulated via a cyclic-AMP dependent protein kinase system and via alpha-adrenergic mechanisms.
J Mol Cell Cardiol 1985 Aug
PMID:The effect of adrenergic agents on the activities of glycerol 3-phosphate acyltransferase and triglyceride lipase in the isolated perfused rat heart. 404 45

Isolated guinea pig hearts were used to determine whether an extracellular (interstitial) or intracellular pool of myocardial adenosine is most important in attenuating the catecholamine-induced enhancement of cardiac contractile state and glycogenolysis. Isoproterenol (2 X 10(-8) M) stimulation of hypoxic (30% O2) perfused hearts produced a marked elevation in tissue and effluent perfusate adenosine levels that were greater than the increases observed with the isoproterenol stimulation of oxygenated hearts (95% O2). In the isoproterenol stimulated hypoxic hearts nitrobenzylthioinosine (NBMPR), a potent inhibitor of adenosine cellular transport, further increased tissue adenosine content and markedly decreased the perfusate level of the nucleoside. Assuming that perfusate levels of adenosine correlate directly with extracellular levels, NBMPR was used as a tool to increase the intracellular and decrease the extracellular content of the nucleoside. When compared to responses in oxygenated hearts, hypoxia reduced the isoproterenol-produced increase in myocardial cyclic AMP content, cyclic AMP-dependent protein kinase activity and contractility but enhanced the increase in glycogen phosphorylase alpha formation. NBMPR completely prevented the reduction of the isoproterenol-induced cyclic AMP and cyclic AMP-dependent protein kinase responses but only partially prevented the attenuation of the contractile response. The increase in phosphorylase alpha formation in the hypoxic isoproterenol stimulated hearts was not influenced by NBMPR. The results suggest that an increase in extracellular adenosine is more influential than an elevation of intracellular adenosine in attenuating beta-adrenoceptor-elicited increases in myocardial cyclic AMP content, cyclic AMP-dependent protein kinase activity and contractile state.
J Mol Cell Cardiol 1984 Sep
PMID:Role of extracellular and intracellular adenosine in the attenuation of catecholamine evoked responses in guinea pig heart. 609 51

To elucidate the role of 22000-dalton protein phospholamban, a putative regulator of Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum, we examined the relationship between cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban and their effects on ATPase activity and calcium transport of cardiac sarcoplasmic reticulum. Cardiac microsomes were incubated with [gamma-32P]ATP or unlabeled ATP, catalytic subunit of cyclic AMP-dependent protein kinase and/or exogenous calmodulin, and subsequently assayed for ATPase activity and calcium uptake by cardiac sarcoplasmic reticulum. Cyclic AMP-dependent phosphorylation of phospholamban was independent of Ca2+, whereas calmodulin-dependent phosphorylation of phospholamban was dependent on Ca2+ within a range between 0.2 and 50 microM. Cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban occurred independently; when both kinases were operative, the amounts of phosphorylation were additive. Under these conditions, the phosphoproteins formed by cyclic AMP- and calmodulin-dependent protein kinases electrophoretically migrated as 11000-dalton components when sodium dodecyl sulfate-solubilized phosphoproteins were boiled prior to polyacrylamide gel electrophoresis. The ATPase activity was stimulated by either cyclic AMP- or calmodulin-dependent phosphorylation of phospholamban at Ca2+ concentrations up to 2 microM. The extents of stimulation of ATPase activity were additive when both types of phosphorylation were functional. Calcium uptake was similarly augmented by cyclic AMP- and/or calmodulin-dependent phosphorylation of phospholamban. These results indicate that Ca2+-dependent ATPase and calcium transport of cardiac sarcoplasmic reticulum are regulated by phospholamban phosphorylation catalyzed by cyclic AMP- and calmodulin-dependent protein kinases, thus suggesting a dual role of phospholamban in active calcium transport.
J Mol Cell Cardiol 1983 May
PMID:Effects of phospholamban phosphorylation catalyzed by adenosine 3':5'-monophosphate- and calmodulin-dependent protein kinases on calcium transport ATPase of cardiac sarcoplasmic reticulum. 631 Jan 31


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