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)

A newly synthesized compound, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), was shown to be a potent inhibitor of two cyclic nucleotide-dependent protein kinases, cyclic GMP-dependent protein kinase and cyclic AMP-dependent protein kinase and the Ki values were 1.4 and 2.3 microM, respectively. HA-1004 relaxed rabbit aortic strips contracted by various agonists and with similar ED50 values. Phenotolamine, propranolol and atropine did not affect this HA-1004-induced relaxation, thereby suggesting that this compound does not act through these membrane receptor associated mechanisms. HA-1004 shifted the dose-response curve for CaCl2 to the right in a competitive manner in depolarized rabbit renal arterial strips. This compound also relaxed the A-23187 and phenylephrine-induced contractions elicited in Ca++-free solution. These findings suggest that HA-1004 exerts its action at the intracellular or submembranal level. This vasodilator has little effect on actomyosin adenosine triphosphatase and Ca++-calmodulin-dependent myosin light chain kinase. Studies using its derivatives with various lengths of alkyl chain (C0-C6) indicated that the potencies of these compounds, as vasorelaxants, correlated well with their potential to inhibit cyclic nucleotide-dependent protein kinase. HA-1004 should be a useful tool for investigating in smooth muscle, regulatory mechanism(s) by second messengers, cyclic AMP and cyclic GMP.
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PMID:Relaxation of vascular smooth muscle by HA-1004, an inhibitor of cyclic nucleotide-dependent protein kinase. 299 36

Cardiac sarcolemma was purified from canine ventricles. Enrichment of the sarcolemmal membranes was demonstrated by the high (Na+ + K+)-ATPase activity of 28.0 +/- 1.5 mumol Pi/mg protein per h and the high concentration of muscarinic receptors with the Bmax of 8.2 +/- 2.5 pmol/mg protein as determined by [3H]QNB binding. The purified sarcolemma also contains significant levels of a membrane-bound Ca2+ and phospholipid-dependent protein kinase (protein kinase C). To elucidate the protein kinase C activity in sarcolemma, a prior incubation of the membranes with EGTA and Triton X-100 was necessary. The specific activity of protein kinase C was found to be 131.4 pmol Pi/mg per min, in the presence of 6.25 micrograms phosphatidylserine and 0.5 mM CaCl2. Treatment of sarcolemma with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBu2) resulted in a concentration-dependent activation of protein kinase C activity. The effect of TPA and PBu2 on protein kinase C in sarcolemma was independent of exogenous Ca2+ and phosphatidylserine. Polymyxin B inhibited phorbol-ester-induced activation of protein kinase C activity. The distribution of protein kinase C in the cytosolic fraction was also examined. The specific activity of the kinase in the cytosolic fraction was 59.7 pmol Pi/mg per min. However, the total protein kinase C activity in the cytosol was 213500 pmol Pi/min, compared to that of 1025 pmol Pi/min in the sarcolemma isolated from approx. 100 g of canine ventricular muscle. Several endogenous proteins in cardiac sarcolemma were phosphorylated in the presence of Ca2+ and phosphatidylserine. The major substrates for protein kinase C were proteins of Mr 94 000, 87 000, 78 000, 51 000, 46 000, 11 500 and 10 000. Most of these substrate proteins have not been identified before. Other proteins of Mr 38 000, 31 000 and 15 000 were markedly phosphorylated in the presence of Ca2+ only. Phosphorylation of phospholamban (Mr 27 000 and 11 000) was also stimulated in the presence of Ca2+ and phosphatidylserine, but the low Mr form of phospholamban was distinct from two other low Mr substrate proteins for protein kinase C. Polymyxin B was more selective in inhibiting the protein kinase C dependent phosphorylation. On the other hand, trifluoperazine selectively inhibited the phosphorylation of phospholamban and Mr 15 000 protein. Although the exact function of this kinase is unknown, based on these observations, we believe that protein kinase C in the cardiac sarcolemma may play an important role in the cell-surface-signal regulated cardiac function.
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PMID:Characterization of the membrane-bound protein kinase C and its substrate proteins in canine cardiac sarcolemma. 308 70

A protein kinase activity phosphorylating regulatory light chain-a (RLC-a) of scallop smooth muscle myosin was found to be present in scallop smooth muscle homogenate. The kinase was purified to homogeneity and named RLC-a myosin kinase (aMK). aMK was extracted from the muscle homogenate with a low salt solution and was purified by successive DE-32 ion exchange chromatography, gel filtration on Ultrogel AcA 44, and affinity chromatography on Sepharose 4B-6-aminohexyl-1-pyrophosphate. The molecular weight of aMK was estimated to be 40-kDa from the mobility on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 35-kDa from the elution volume on Sephadex G-150 gel filtration. The phosphorylation site of RLC-a by aMK was determined to be Ser residue(s). Only RLC-a was phosphorylated; the other regulatory light chain, RLC-b, was not. The phosphorylatable Ser of RLC-a is, therefore, considered to be Ser-11, which is located in the N-terminal region having a different amino acid sequence from that of RLC-b. RLC-a was phosphorylated by aMK 3 times faster in the free state than in the bound state to myosin. aMK does not require calmodulin and is rather inhibited by CaCl2.
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PMID:Purification of a protein kinase phosphorylating myosin regulatory light chain-a (RLC-a) from smooth muscle of scallop, Patinopecten yessoensis. 310 65

Stimulatory effects of Ca2+-CaM and PKI on partially purified hypothalamic HD (10 fold purification) have been shown under conditions involving inhibition of the enzyme by cAMP-induced phosphorylation and under control conditions. A 1:1 (v/v) mixture of 0.1 mM CaCl2 and 10 units of CaM from human red blood cells reversed the inhibition of HD induced by cAMP-dependent protein phosphorylation activity to the control level. Verapamil (0.01 mM) could partially block the former effect without affecting the control level of enzyme activity. 0.01 mM TPA did not further increase the effect of Ca2+-CaM on HD, in the presence of 0.01 mM ATP, indicating that this stimulation does not require the action of Ca2+-dependent protein kinase. The control level of HD is not influenced by 0.1 mM CaCl2 or 0.02 mM EGTA but is raised by CaM in the presence of CaCl2 (0.1 mM). A highly purified protein kinase (cAMP-dependent) inhibitor (PKI) from bovine heart and a crude inhibitor from rat cerebellum could also reverse the inhibitory effect of cAMP-dependent protein kinase under phosphorylating conditions and enhanced HD activity above control levels. PKI and Ca2+-CaM, added together, produced single, not additive effects. We conclude that cAMP-induced phosphorylation is probable the main regulatory mechanism of histamine formation and this could be influenced by both Ca2+-CaM and PKI. Inhibition of cAMP-dependent protein kinase as well as stimulation of phosphoprotein phosphatase and Ca2+-CaM-dependent phosphodiesterase might be involved in the above actions.
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PMID:Stimulation of hypothalamic histidine decarboxylase by calcium-calmodulin and protein kinase (cAMP-dependent) inhibitor. 360 3

A study was performed to determine whether the proteins phosphorylated by cAMP dependent and calmodulin dependent protein kinase in vascular smooth muscle membrane fractions represent integral membrane proteins or are tightly associated cytoskeletal proteins. In the unextracted microsomal fraction cAMP dependent protein kinase phosphorylated proteins of 240K, 105K, and 48K daltons. Similarly, calmodulin dependent protein kinase phosphorylated 65K, 60K, 48K, and 20K dalton bands. The 48K dalton band represented a major protein in the microsomal fraction, and it was a common substrate for both cAMP dependent and calmodulin dependent protein kinase, and the extent of phosphorylation by two kinases was additive. The 48K dalton band showed immunological reaction with monoclonal antibodies raised against human umbilical artery F actin, and it also comigrated with arterial smooth muscle actin on SDS gel electrophoresis. Plasma membranes prepared from vascular smooth muscle microsomes after extraction with actomyosin extraction buffer consisting of 2 mmol X litre-1 TRIS-maleate, pH 6.8, 0.25 mol X litre-1 sucrose, 0.5 mmol X litre-1 ATP, 0.2 mmol X litre-1 CaCl2, 0.1 mmol X litre-1 phenylmethylsulphonylfluoride, and 1 mmol X litre-1 dithiothreitol yielded membranes that were substantially free from cytoskeletal protein contamination. These membranes were enriched 60-80 fold in plasma membrane marker enzymes and showed energy dependent calcium uptake. In the extracted plasma membranes none of the proteins was phosphorylated by cAMP dependent or calmodulin dependent protein kinase. Thus these results show that protein phosphorylated in the unextracted microsomal fraction or unextracted plasma membranes are not integral plasma membrane proteins but represent tightly associated cytoskeletal proteins.
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PMID:Phosphorylation of cytoskeletal proteins tightly associated with the vascular smooth muscle membranes. 371 12

"Heavy" sarcoplasmic reticulum vesicles loaded with 5 mM CaCl2 in the presence of protease inhibitors were phosphorylated by addition of MgATP in the presence or absence of calmodulin. The major site of phosphorylation was a 60-kDa protein. In the absence of added calmodulin, phosphorylation of the 60-kDa protein reached its maximal value (8 pmol of P/mg of membrane protein) at 1 min. In the presence of 1 microM calmodulin, a 2-fold higher level of phosphorylation (16.1 pmol of P/mg of sarcoplasmic reticulum) was reached within a shorter time (10 s). The phosphoprotein was then spontaneously dephosphorylated. The initial rate of Ca2+ release, which was induced by a Ca2+ jump and determined by stopped-flow fluorometry using chlorotetracycline, decreased upon phosphorylation, whereas it was restored upon dephosphorylation. There was good correlation between the amount of P incorporation into the 60-kDa protein and the extent of inhibition of Ca2+ release. In the presence of added calmodulin the protein kinase activity sharply increased in the [Ca2+] range of 0.2-2 microM with a concentration for half-maximal activation at 0.6 microM. On the other hand, the protein phosphatase activity was virtually independent of calmodulin and [Ca2+] in the [Ca2+] range in which protein kinase was activated. The results suggest that the calmodulin-dependent phosphorylation of the 60-kDa protein plays an important role in the regulation of Ca2+ release from sarcoplasmic reticulum.
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PMID:Involvement of 60-kilodalton phosphoprotein in the regulation of calcium release from skeletal muscle sarcoplasmic reticulum. 374 63

Competition experiments using 9-anthroylcholine, a fluorescent dye that undergoes calmodulin-dependent binding by smooth muscle myosin light chain kinase [Malencik, D. A., Anderson, S. R., Bohnert, J. L., & Shalitin, Y. S. (1982) Biochemistry 21, 4031], demonstrate a strongly stabilizing interaction between the adenosine 5'-triphosphate and myosin light chain binding sites operating within the enzyme-calmodulin complex but probably not in the free enzyme. The interactions in the latter case may be even slightly destabilizing. The fluorescence enhancement in solutions containing 5.0 microM each of the enzyme and calmodulin is directly proportional to the maximum possible concentration of bound calcium on the basis of four calcium binding sites. Evidently, all four calcium binding sites of calmodulin contribute about equally to the enhanced binding of 9-anthroylcholine by the enzyme. Fluorescence titrations on solutions containing 1.0 microM enzyme plus calmodulin yield a Hill coefficient of 1.2 and K = 0.35 +/- 0.08 microM calcium. Three proteolytic fragments of smooth muscle myosin light chain kinase, apparent products of endogenous proteolysis, were isolated and characterized. All three possess calmodulin-dependent catalytic activity. Their interactions with 9-anthroylcholine, in both the presence and absence of calmodulin, are similar to those of the native enzyme. However, the stabilities of their complexes with calmodulin vary. The corresponding dissociation constants range from 2.8 nM for the native enzyme and 8.5 nM for the 96K fragment to approximately 15 nM for the 68K and 90K fragments [0.20 N KCl, 50 mM 3-(N-morpholino)propanesulfonic acid, and 1 mM CaCl2, pH 7.3, 25 degrees C]. A coupled fluorometric assay, modified from a spectrophotometric assay for adenosine cyclic 3',5'-phosphate dependent protein kinase [Cook, P. F., Neville, M. E., Vrana, K. E., Hartl, F. T., & Roskoski, R. (1982) Biochemistry 21, 5794], has provided the first continuous recordings of myosin light chain kinase phosphotransferase activity. The results show that smooth muscle myosin light chain kinase is a responsive enzyme, whose activity adjusts rapidly to changes in solution conditions.
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PMID:Calmodulin-linked equilibria in smooth muscle myosin light chain kinase. 375 54

This investigation was carried out to determine if calcium prolongation of ethanol-induced sleep is mediated by calmodulin and a calmodulin-dependent protein kinase. The duration of ethanol-induced sleeping time in ddY male mice was measured following the administration of CaCl2 (20, 40, 80 and 200 mumol/kg, intraperitoneally (IP) both with and without the calmodulin antagonists, W-7: [N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide] (4.2 micrograms/mouse, intraventricular (IVT) or trifluoperazine (TFP; 1.8 micrograms/mouse, IVT). When CaCl2 was administered in a dose dependent manner the duration of ethanol-induced sleep was prolonged. The prolongation was antagonized by W-7 and TFP. When mice were treated with W-7 or TFP together with serotonin (5-HT; 15 nmol/mouse, IVT), dopamine (DA; 30 nmol/mouse, IVT) or norepinephrine (NE; 30 nmol/mouse, IVT), the sleeping time induced by ethanol and calcium was enhanced. This finding suggests that W-7 and TFP selectively inhibit the synthesis of 5-HT, DA and NE, but they do not affect other neuronal functions of these biogenic amines. The results would suggest a probable mechanism in which Ca++ prolongs ethanol-induced sleeping time by activating tyrosine hydroxylase and tryptophan hydroxylase through intracerebral calmodulin and calmodulin-dependent protein kinase, which subsequently raise the levels of 5-HT, DA and NE.
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PMID:Effect of calmodulin antagonists on calcium and ethanol-induced sleeping time in mice. 407 Mar 38

The pyruvate dehydrogenase complex has been demonstrated in high speed pellet preparations from sonicated ribbed mussel gill mitochondria. The activity of the complex is inhibited by low chloride (less than 100 mM) concentrations, EDTA (1 mM), succinate, ATP, and NAD/NADH ratios below 4. Inhibition by EDTA is relieved by addition of 10 mM MgCl2-1 mM CaCl2. ATP inhibition was enhanced by NaF and reversed by high Mg++ concentrations in the absence of NaF. Pyruvate and thiamine pyrophosphate inhibited the inactivation by ATP. The nonhydrolyzable ATP analog AMP-PNP caused inhibition of the overall catalytic activity that was identical to ATP. Factors involved in the ATP inhibition and Mg++ reversal are lost with freezing or cold storage. Preliminary results using gamma-32P-ATP indicate that a protein kinase that phosphorylates the alpha subunit of E1 (pyruvate dehydrogenase) from the mammalian PDC is associated with the gill PDC. The activity of the complex may be regulated by a phosphorylation/dephosphorylation mechanism and by the relative levels of substrates, products, and other metabolites in the mitochondria.
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PMID:Pyruvate dehydrogenase complex from ribbed mussel gill mitochondria. 408 84

Activation of rat striatal tyrosine hydroxylase [TyrOHase; tyrosine monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] by ATP/Mg2+ and endogenous protein kinase can be produced without the addition of cAMP. This activation is not due to endogenous free catalytic subunit derived from cAMP-dependent protein kinase. In the presence of amounts of protein kinase inhibitor sufficient for complete inhibition of striatal cAMP-dependent protein kinase and the cAMP-mediated activation of TyrOHase, addition of ATP/Mg2+ results in an enhancement of TyrOHase activity. Enzyme activation does not occur when the nonhydrolyzable form of ATP, adenylyl imidodiphosphate, is substituted for ATP. When TyrOHase is assayed in the presence of ATP/Mg2+ and different concentrations of either tyrosine or 6-methyltetrahydropterin co-factor, a 2-fold increase in enzyme Vmax is demonstrable, with no change in the Km for either substrate or cofactor. In contrast, in the presence of cAMP and ATP/Mg2+, both an increase in Vmax and an enhanced affinity for pterin cofactor are demonstrable. In the latter circumstance, the 2-fold increase in Vmax can be attributed entirely to the action of cAMP-independent protein kinase. The addition of either EGTA or CaCl2 does not modify the effect seen in the presence of ATP, suggesting that the effect of ATP/Mg2+ is not mediated by a Ca2+-dependent protein kinase. These data support the existence of a cAMP-independent striatal protein kinase that can catalyze the activation of TyrOHase.
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PMID:Evidence for the involvement of a cyclic AMP-independent protein kinase in the activation of soluble tyrosine hydroxylase from rat striatum. 613 85


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