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 studied the compartmentation of cyclic AMP action in purified ventricular cardiomyocytes prepared by collagenase perfusion of adult rabbit hearts. Incubation of purified adult myocytes with 1 microM isoproterenol causes rapid accumulation of intracellular cyclic AMP in both soluble (2.3 leads to 7.7 pmol/ mg of protein) and particulate (3.0 leads to 9.2) fractions of cell homogenates (3000 X g for 5 min), increases in the total activity and activity ratio of soluble cyclic AMP-dependent protein kinase (0.21 leads to 0.66), a decrease in protein kinase activity remaining in the particulate fraction (47 leads to 30%), and an increase in the activity ratio of glycogen phosphorylase (0.15 leads to 0.47). Incubation of myocytes with 10 microM prostaglandin E1 (PGE1) leads to a comparable increase in soluble cyclic AMP (2.3 leads to 5.8 pmol/mg of protein) and activation of soluble cyclic AMP-dependent protein kinase (0.21 leads to 0.39) but does not result in any change in cAMP or protein kinase in the particulate fraction and fails to cause an activation of glycogen phosphorylase. PGE1 does not inhibit the effects of isoproterenol; when myocytes are incubated with both isoproterenol and PGE1, the accumulation of cyclic AMP, activation of cAMP-dependent protein kinase and phosphorylase b leads to a conversion are equal to that achieved with isoproterenol alone. Perturbation of cellular calcium using the ionophore A23187, verapamil, or high or low extracellular calcium did not alter the ability of isoproterenol to cause activation of particulate cAMP-dependent protein kinase or influence the inability of PGE1 to do so. Activation of adenylate cyclase by forskolin (30 microM) caused immediate activation of both soluble and particulate cAMP-dependent protein kinase leading to rapid activation of phosphorylase. We conclude that the hormonally specific compartmentation of cyclic AMP and cAMP-dependent protein kinase that occurs in intact heart (Hayes, J. S., Brunton, L. L., Brown, J. H., Reese, J. B., and Mayer, S. E. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1570-1574) is not explained on the basis of cellular heterogeneity but has a subcellular basis within the cardiomyocyte.
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PMID:Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. 630 96

A calmodulin-dependent glycogen synthase kinase distinct from phosphorylase kinase has been purified approximately equal to 5000-fold from rabbit skeletal muscle by a procedure involving fractionation with ammonium sulphate (0-33%), and chromatographies on phosphocellulose, calmodulin-Sepharose and DEAE-Sepharose. 0.75 mg of protein was obtained from 5000 g of muscle within 4 days, corresponding to a yield of approximately equal to 3%. The Km for glycogen synthase was 3.0 microM and the V 1.6-2.0 mumol min-1 mg-1. The purified enzyme showed a major protein staining band (Mr 58 000) and a minor component (Mr 54 000) when examined by dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was determined to be 696 000 by sedimentation equilibrium centrifugation, indicating a dodecameric structure. Electron microscopy suggested that the 12 subunits were arranged as two hexameric rings stacked one upon the other. Following incubation with Mg-ATP and Ca2+-calmodulin, the purified protein kinase underwent an 'autophosphorylation reaction'. The reaction reached a plateau when approximately equal to 5 mol of phosphate had been incorporated per 58 000-Mr subunit. Both the 58 000-Mr and 54 000-Mr species were phosphorylated to a similar extent. Autophosphorylation did not affect the catalytic activity. The calmodulin-dependent protein kinase initially phosphorylated glycogen synthase at site-2, followed by a slower phosphorylation of site-1 b. The protein kinase also phosphorylated smooth muscle myosin light chains, histone H1, acetyl-CoA carboxylase and ATP-citrate lyase. These findings suggest that the calmodulin-dependent glycogen synthase kinase may be a enzyme of broad specificity in vivo. Glycogen synthase kinase-4 is an enzyme that resembles the calmodulin-dependent glycogen synthase kinase in phosphorylating glycogen synthase (at site-2), but not glycogen phosphorylase. Glycogen synthase kinase-4 was unable to phosphorylate any of the other proteins phosphorylated by the calmodulin-dependent glycogen synthase kinase, nor could it phosphorylate site 1 b of glycogen synthase. The results demonstrate that glycogen synthase kinase-4 is not a proteolytic fragment of the calmodulin-dependent glycogen synthase kinase, that has lost its ability to be regulated by Ca2+-calmodulin.
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PMID:The calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle. Purification, subunit structure and substrate specificity. 631 30

A hypersensitivity of glycogen phosphorylase activation by epinephrine and glucagon has been demonstrated in isolated perfused working and non-working hearts from diabetic rats. Accumulation of tissue cAMP and activation of cAMP-dependent protein kinase in response to epinephrine and glucagon were no greater and usually less in hearts of diabetic than of normal rats. Insulin deficiency was not associated with greater changes in epinephrine-induced activation of glycogen phosphorylase kinase than that observed in normal hearts. Perfusion of hearts with subphysiological concentrations of calcium (0.83 mM) partially reversed the diabetes-related hypersensitivity of phosphorylase activation by epinephrine. The phosphorylase activation hypersensitivity to epinephrine was completely reversed by adrenalectomizing diabetic rats 5 days before heart perfusion, an effect potentially caused by steroid-induced changes in cardiac calcium metabolism. These data are consistent with the hypothesis that phosphorylase activation by phosphorylase kinase is allosterically increased in the diabetic due to a diabetes-related increase in free intracellular calcium concentrations.
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PMID:Phosphorylase activation hypersensitivity in hearts of diabetic rats. 632 Jun 71

The following article provides evidence that cellular calcium controls the activity of glycogen synthase in all three major glycogen storage tissues; muscle, fat, and liver. Depletion of cellular calcium resulted in a moderate increase of glycogen synthase %I activities in intact mouse diaphragms, in isolated rat adipocytes, and in rat hepatocytes. The increase in %I activity of glycogen synthase was more pronounced when the uridine di-phosphoglucose concentration in the glycogen synthase assay was lowered from 4.4 mM to 0.2 mM. Calcium depletion resulted in an approximately two-fold decrease in the Ka values for glucose-6-phosphate in all three tissues. The activities of glycogen synthase also correlated well with the content of cell-associated calcium in rat hepatocytes. The glucose-6-phosphate independent activities of glycogen synthase in extracts of calcium-replete and calcium-depleted tissue approached the same value following the exposure to crude phosphoprotein phosphatase. The activities of glycogen phosphorylase decreased in calcium-depleted tissues and cells. Insulin stimulated the activity of glycogen synthase in muscle and fat in the absence of added sugar and in the absence of extracellular calcium. It is concluded that glycogen synthase is under the control of calcium in the three main glycogen storage tissues. The actions of calcium are probably mediated through the actions of calcium-sensitive protein kinase(s).
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PMID:Calcium control of glycogen synthase activities in mouse diaphragms, rat adipocytes and rat hepatocytes. 642 46

Incubation of cytosol fractions from a variety of mammalian tissues (heart, liver, lung, adrenal, spleen and skeletal muscle) with Ca2+ (0.5 mM) in the presence of gamma-[32P]ATP resulted in the phosphorylation of a prominent substrate of Mr approximately 100 000 (100 kDa). One-dimensional peptide maps and two-dimensional tryptic fingerprints of the phosphoprotein from these sources were identical. A single major phosphopeptide was generated by trypsin and was determined to contain exclusively phosphothreonine. The 100 kDa substrate could be distinguished from glycogen phosphorylase (Mr approximately 97 000) by a number of criteria including phosphopeptide mapping and by its failure to bind either to glycogen or to a specific antiphosphorylase antibody. The Ca2+-dependent protein kinase responsible for phosphorylation of the 100 kDa protein appeared to be a calmodulin (CaM)-requiring enzyme in that it could be inhibited in cytosol extracts by trifluoperazine (IC50 6-16 microM) and that exogenous CaM was necessary for 100 kDa phosphorylation in CaM-depleted cytosol. These results suggest that a rise in intracellular Ca2+ resulting in an activation of CaM-dependent protein kinase leads to the phosphorylation of a common 100 kDa substrate in many tissues.
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PMID:Presence in many mammalian tissues of an identical major cytosolic substrate (Mr 100 000) for calmodulin-dependent protein kinase. 686 15

Rats from an inbred strain (NZR/Mh) were found to have high concentrations of glycogen in their livers, even after 24 h of starvation. Despite this, blood glucose concentrations were well maintained on starvation for up to 72 h. The primary defect is a deficiency of liver phosphorylase kinase, causing a lack of active glycogen phosphorylase, although total phosphorylase is normal. The intravenous injection of glucagon caused a rapid activation of cyclic AMP-dependent protein kinase in the liver, but no increase in either phosphorylase kinase or phosphorylase a activity. Although total glycogen synthase activity in the livers of affected rats was higher than normal, glycogen synthase in the active form was very low, presumably as a result of the high liver glycogen content. The condition is transmitted as autosomal recessive and, apart from hepatomegaly, the affected rats appear healthy.
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PMID:Glycogen-storage disease in rats, a genetically determined deficiency of liver phosphorylase kinase. 693 96

Freshwater turtles Trachemys scripta elegans endure prolonged severe hypoxia, and even complete anoxia, while diving or hibernating underwater. Metabolic adaptations supporting survival include the activation of glycogenolysis and glucose output from liver, as well as strong metabolic rate depression. The present study analyzes the enzymes of both the phosphorolytic (glycogen phosphorylase, phosphorylase b kinase, cAMP-dependent protein kinase) and glucosidic (alpha-glucosidase) pathways of glycogenolysis in turtle organs. Turtles were subjected to 5 hr of submergence in N2-bubbled water at 7 degrees C and then activities of phosphorolytic and glucosidic enzymes were assayed in liver, heart, brain, and red and white skeletal muscle, and compared with aerobic controls. In vitro incubations also assessed protein kinase A control of phosphorolytic enzymes. A functional enzyme cascade system for the activation of glycogen phosphorylase was found in all organs, and both phosphorylase and phosphorylase kinase were stimulated by in vitro incubation with the catalytic subunit of cAMP-dependent protein kinase. Anoxic submergence led to significant increases in phosphorylase activities in liver and heart (phosphorylase a rose 2- and 2.5-fold, respectively) but phosphorylase kinase and protein kinase A activities in liver were reduced after 5 hr exposure. Both acidic (pH 4) and neutral (pH 7) forms of alpha-glucosidase were detected in all five organs with highest activities in liver. Activity of acid alpha-glucosidase, which degrades lysosomal glycogen, increased by 2-fold in liver during anoxic submergence. The data show that glycogen breakdown in turtle liver during anoxic submergence may result from coordinated activations of both the cytoplasmic phosphorolytic and the lysosomal glucosidic pathways of glycogenolysis.
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PMID:Enzymatic control of glycogenolysis during anoxic submergence in the freshwater turtle Trachemys scripta. 758 17

The mechanism of yeast glycogen phosphorylase activation by covalent phosphorylation involves structural elements distinct from the mammalian homologs. To understand the role of the amino-terminal 39-residue extension in the phosphorylation control mechanism, mutants with 22 and 42 amino-terminal residues removed were expressed in Escherichia coli, and their properties were compared with the wild-type (WT) enzyme. The unphosphorylated WT enzyme had a specific activity of 0.1 unit/mg and was not activated significantly by the substrate, glucose 1-phosphate. Phosphorylation by protein kinase resulted in a 1300-fold activation. Glucose 6-phosphate inhibited the unphosphorylated enzyme more effectively than the phosphorylated form, and inhibition of the latter was cooperative. Glucose was a poor inhibitor for both the unphosphorylated and phosphorylated WT enzyme with Ki > 300 mM. The rate of phosphorylation by protein kinase depended on substrates and interactions of the amino terminus. Maltoheptaose increased the rate of phosphorylation of the WT enzyme by yeast phosphorylase kinase 5-fold. The 22-residue deletion mutant (Nd22) had overall kinetic properties similar to the WT enzyme, except that Nd22 was a better substrate for the protein kinase and the rate of phosphorylation was unaffected by maltoheptaose. The 42-residue deletion mutant (Nd42), which lacks the phosphorylation site, was measurably active, although much less active than phosphorylated WT. Sedimentation equilibrium analysis indicated that the WT, Nd22, and Nd42 exist as tetramer, partially dissociated tetramer, and dimer, respectively. Phosphorylation of the WT and Nd22 converted both to dimer. The results indicated that the amino terminus affects quaternary structure and mediates activity regulation through conformational transition.
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PMID:Mechanism of regulation in yeast glycogen phosphorylase. 759 25

The Saccharomyces cerevisiae gene PPZ1 codes for a 692-residues protein that shows in its carboxyl-terminal half about 60% identity with the catalytic subunit of mammalian and yeast protein phosphatase-1 and that is involved in salt homeostasis. The complete PPZ1 protein has been successfully expressed as a soluble glutathione-S-transferase fusion protein. The recombinant protein, after purification by a single affinity chromatography step, displayed phosphatase activity towards a number of substrates, including myelin basic protein, histone 2A and casein, but was ineffective in dephosphorylating glycogen phosphorylase. It was also active towards p-nitrophenylphosphate. The activity was severalfold increased by the presence of Mn2+ ions and by limited trypsinolysis. The enzyme was inhibited by okadaic acid and microcystin-LR at concentrations comparable to what is found for type 1 protein phosphatase although it was much less sensitive to inhibitor-2. The recombinant protein was phosphorylated in vitro by cAMP-dependent protein kinase, protein kinase C and casein kinase-2. Phosphorylation affected preferentially sites located in the amino-terminal half of the protein and did not alter the activity of the phosphatase.
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PMID:Biochemical characterization of recombinant yeast PPZ1, a protein phosphatase involved in salt tolerance. 761 85

Organ slices from the turtle Trachemys scripta elegans were incubated under aerobic and anoxic conditions to examine the effect of protein kinase (PrK) second messengers in potentiating the biochemical responses to anoxia exposure. Incubating liver slices from aerobic animals under anoxic conditions produced biochemical changes exactly similar to those observed in vivo: phosphofructokinase (PFK) was more sensitive to citrate inhibition and the percentage of glycogen phosphorylase (GP) in the active a form increased. On the other hand, incubating brain and heart tissue slices under anoxic conditions produced no changes in PFK and GP kinetic constants. Addition of PrK second messengers (dibutyryl-cAMP or Ca2+ plus phorbol myristate acetate) to the incubated tissues did not promote anoxia-associated changes in aerobically incubated tissues nor did they prevent anoxia-associated changes in anaerobically incubated tissues. These results suggest that unidentified external hormonal signals mediate heart and brain responses to anoxia. It is also apparent that cAMP and Ca2+ plus phospholipid do not play a role in bringing about the anoxia-induced changes in PFK, GP and fructose 2,6-bisphosphate in liver of turtles.
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PMID:Effect of anoxia on isolated turtle tissues: is the response to anoxia mediated by protein kinase second messengers? 769 98


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