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:3.6.1.3 (ATPase)
65,361 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

The prevailing conformations of partially purified pig kidney (Na+ + K+)-ATPase interacting with ligands related to its phosphatase activity were determined following time-dependent trypsin digestion and inactivation as well as the amounts of Rb+ or Ca2+ bound to the enzyme after passage through cation-exchange resin columns. In the presence of 150 mM choline chloride, alone or with 3 mM MgCl2, 3 mM MnCl2 or 1 mM CaCl2, the major enzyme conformation was E1. Similar forms were seen with 5 mM p-nitrophenyl phosphate with and without 3 mM MgCl2. KCl, at 0.5 mM or 150 mM, produced an E2 enzyme state; the effects of 0.5 mM KCl were completely counteracted by 5 mM p-nitrophenyl phosphate. Under optimal conditions for phosphatase activity (3 mM MgCL2/5 mM p-nitrophenyl phosphate/10 mM KCl) the (Na+ + K+)-ATPase was in the E2 state. At low ionic strength and 20 degrees C and under 85% of maximal RbCl-stimulated phosphatase turnover (1 mM RbCl/3 mM MgCl2/5 mM p-nitrophenyl phosphate) no Rb+ occlusion could be detected. Ca2+, at low ionic strength and in the presence of 3 mM MgCl2, stimulated an ouabain-sensitive phosphatase activity. The rates of hydrolysis obtained wit 1 mM CaCl2 were similar to those seen with 0.5 mM KCl; under both conditions, similar patterns of trypsin digestion and inactivation of the enzyme were obtained. On the other hand, Ca2+ could not mimic Rb+ in its ability to induce an E2-occluding state. These results suggest that during phosphatase activity of (Na+ + K+)-ATPase, the most abundant form is a non-occluding E2 and that at least one of the mechanisms of potassium stimulation of that activity it to take the enzyme into the E2 state.
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PMID:Phosphatase activity of (Na+ + K+)-ATPase. Ligand interactions and related enzyme forms. 299 63

Brush-border membranes were isolated from rabbit small intestine by procedures involving precipitation of undesired membranes with either 10 mM MgCl2 or 10 mM CaCl2. The membranes were compared on the basis of marker enzyme content and lipid composition. Ca2+-prepared membranes displayed a greater enrichment of alkaline phosphatase and sucrase activity compared to homogenate than did the Mg2+-prepared membranes. The former also displayed an impoverishment of (Na+ + K+)-ATPase activity, the specific activity of which increased several-fold in Mg2+-prepared membranes. Membranes prepared with Ca2+ were characterized by a lower phosphoacylglycerol-protein ratio and a higher phosphatidylethanolamine-phosphatidylcholine ratio. Although lysophosphoacylglycerols accounted for about 6% of the total phospholipids in these membranes compared to 2% in Mg2+-prepared membranes, the free fatty acid content was similar in both types of membranes. It was concluded that Ca2+ prepared membranes were less contaminated by basolateral membranes than were Mg2+-prepared membranes and the use of Ca2+ did not notably enhance degradation of endogenous lipids by brush-border membrane phospholipase A.
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PMID:A comparison of brush-border membranes prepared from rabbit small intestine by procedures involving Ca2+ and Mg2+ precipitation. 300 39

The effect of the local intra-arterial infusion of ouabain (11.8 micrograms/min.) on the response of the forelimb to vasoactive agents was examined. In seven dogs, bolus injections of CaCl2, MgSO4, KCl, norepinephrine, adenosine, acetylcholine, PGE1 and saline were made into the forelimb perfused at constant flow before and three times during ouabain infusion. Ouabain blocked potassium vasodilation and changed the response to CaCl2 from vasoconstriction to vasodilation. The response of the forelimb to the other vasoactive agents was initially unaffected by ouabain but with time the forelimb vasculature became less sensitive to all agents studied. These changes were not seen in a series of 5 saline infused control animals. In a third series of animals steady-state dose responses to CaCl2, Ca-gluconate and KCl were explored by infusing solutions intrabrachially at three dosages. Before ouabain, forelimb resistance increased as a function of Ca++ and decreased as a function of K+. Ouabain completely blocked potassium vasodilation and on the average blocked Ca++ vasoconstriction although a number of animals evidenced vasodilation to Ca++ during ouabain infusion. These data indicate that K+ vasodilation is Na+, K+-ATPase dependent and that Na+, K+-ATPase inhibition unmasks a vasodilatory action of locally applied Ca++.
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PMID:Response of the forelimb vasculature to vasoactive agents: effects of ouabain. 301 99

1. Purealin, a novel bioactive principle of a sea sponge Psammaplysilla purea, activated the superprecipitation of myosin B (natural actomyosin) from rabbit skeletal muscle. The maximum change in the turbidity increased with increasing purealin concentrations and was three times the control value in the presence of 50 microM purealin. 2. The ATPase activity of myosin B was also elevated to 160% of the control value by 10 microM purealin. On the other hand, purealin inhibited the myosin ATPase in the presence of 10 mM CaCl2 and 0.5 M KCl (Ca2+-ATPase), and the concentration for the half inhibition was 4 microM. 3. On the other hand, purealin activated the myosin ATPase in the presence of 5 mM EDTA and 0.5 M KCl (EDTA-ATPase). The maximum activation by 10 microM purealin was 160% of the control value. 4. Furthermore, similar results concerning the modification of ATPase activities by purealin were obtained in myosin subfragment-1 instead of myosin. 5. These results suggest that purealin activates the superprecipitation of myosin B by affecting the myosin heads directly. It is also an interesting observation that there is a correlation between the activities of the myosin EDTA-ATPase and actomyosin ATPase of myosin B.
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PMID:Purealin, a novel activator of skeletal muscle actomyosin ATPase and myosin EDTA-ATPase that enhanced the superprecipitation of actomyosin. 304 Mar 94

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

We have investigated whether muscarinic receptors modulate the release of [3H]ACh elicited by secretagogues that act by different mechanisms in rat cerebral cortical synaptosomes. Oxotremorine (10 microM) reduced the calcium-dependent [3H]ACh release induced by mild K+-depolarization (10 and 15 mM K+), but not that by higher K+ concentrations. The ACh-release induced by A23187 (0.2-5 micrograms/ml), liposomes laden with 113 mM CaCl2, or 4-aminopyridine (1-10 mM) was not modulated by oxotremorine. Ouabain (100 microM)-induced release of [3H]ACh was reduced by oxotremorine in normal but not calcium-free KR, indicating that extracellular calcium-uptake but not Na+, K+-ATPase activity may be necessary for release-modulation. With respect to possible second messenger systems, dibutyrylcyclic AMP (0.1-2 mM), dibutyrylcyclic GMP (0.1-2 mM), forskolin (100 microM), and phorbol ester (0.3-3 micrograms/ml) were without effect on release or release-modulation. These results are consistent with an involvement of K+-channels and voltage-sensitive calcium-channels in the muscarinic release-inhibition process. They argue against an involvement of Na+, K+-ATPase, adenylate cyclase, guanylate cyclase, and phosphatidylinositol turnover in the release-modulation process.
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PMID:Effects of different secretagogues and intracellular messengers on the muscarinic modulation of [3H]acetylcholine release. 312 25

H+ and Ca2+ concentration changes in the reaction medium following MgATP addition at pH 6.0 were determined with the partially purified Ca-ATPase from sarcoplasmic reticulum vesicles in the presence of 25-50 microM CaCl2 and 5 mM MgCl2 at 4 degrees C. Previously, we showed a sequential occurrence of H+ binding and H+ dissociation in the Ca-ATPase during ATP hydrolysis and further suggested that the H+ binding takes place inside the vesicles (Yamaguchi, M., and Kanazawa, T. (1984) J. Biol. Chem. 259, 9526-9531). The present results demonstrate that the H+ binding occurred coincidently with Ca2+ dissociation from the enzyme upon conversion of the phosphoenzyme (EP) intermediate from the ADP-sensitive form to the ADP-insensitive form in the catalytic cycle of ATP hydrolysis. As KCl decreased in the medium, the extent of the H+ binding increased almost proportionately with the extent of either the Ca2+ dissociation or the accumulation of ADP-insensitive EP. Both the H+ binding and the Ca2+ dissociation were prevented by a modification of the specific SH group of the enzyme essential for the conversion of ADP-sensitive EP to ADP-insensitive EP. In the late stage of the reaction, H+ dissociation from the enzyme occurred coincidently with Ca2+ binding to the dephosphoenzyme which was formed by EP decomposition. These results are consistent with the possibility that the H+ ejection during the Ca2+ uptake with the intact vesicles previously shown by several investigators takes place through a Ca2+/H+ exchange directly mediated by the membrane-bound Ca-ATPase.
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PMID:Coincidence of H+ binding and Ca2+ dissociation in the sarcoplasmic reticulum Ca-ATPase during ATP hydrolysis. 315 86

The Ca-ATPase of sarcoplasmic reticulum was solubilized at pH 6.5 and 30 degrees C using different nonionic detergents, Triton X-100, C12E8, Lubrol PX, or Tween 20. After full solubilization by any of these detergents, the enzyme was unstable (t1/2 = 2-3 min) in the absence of Ca2+. The soluble enzyme was stable in the presence of calcium, half-maximal protection being attained in the presence of 0.2 mM Ca2+. In the absence of Ca2+, stability was restored by addition of co-solvents dimethyl sulfoxide or glycerol. In the presence of 4 mM Ca2+, the progressive addition of nonionic detergents to a medium containing leaky vesicles promoted an increase, up to 3-fold, in the rate of ATP hydrolysis. This was not observed when ITP was used as substrate. The small amount of ADP accumulated in the medium during ATP hydrolysis was sufficient to inhibit the ATPase activity of the membrane-bound enzyme but had no effect on the soluble enzyme. Increasing concentrations of detergent promoted a progressive inhibition of the ATP----Pi exchange reaction. The ATP hydrolysis/synthesis ratio of soluble enzyme was 10 times higher than that of membranous enzyme. Addition of co-solvent restored this ratio to values similar to those obtained with membrane-bound Ca-ATPase. Soluble enzyme prepared from native sarcoplasmic reticulum vesicles was able to catalyze the net synthesis of ATP when phosphorylated by Pi in the presence of dimethyl sulfoxide and then diluted in a medium containing 10 mM CaCl2 and 2 mM ADP. This was not observed when the soluble enzyme was prepared from purified Ca-ATPase. The results suggest that some of the partial reactions of the catalytic cycle of Ca-ATPase are dependent on the hydrophobic environment found in the native membrane. This environment can be mimicked by co-solvents.
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PMID:Stability and partial reactions of soluble and membrane-bound sarcoplasmic reticulum ATPase. 315 50

The effects of intra- and extravesicular calcium and magnesium ions on the hydrolysis of the phosphoenzyme (EP) intermediate formed in the reaction of Ca2+,Mg2+-dependent ATPase of the sarcoplasmic reticulum were investigated. The rate constants of EP hydrolysis were measured under conditions that allowed a single turnover of ATP hydrolysis to minimize the increase in calcium concentration inside the vesicles. The EP formed during a single turnover was hydrolyzed biphasically and could be resolved into fast- and slow-decomposing components. When free Mg2+ outside the vesicles was chelated by adding excess EDTA, EP could also be kinetically resolved into two components; EDTA-sensitive EP, which could be quickly decomposed by adding EDTA, and EDTA-insensitive EP, which could be prevented from decomposing by adding EDTA. The amount of EDTA-sensitive EP decreased rapidly during the initial phase of the reaction, while that of EDTA-insensitive EP decreased slowly with the same rate constant as that of the slow-decomposing EP. These results showed that the biphasic time course of EP hydrolysis was caused by the formation of EDTA-sensitive and -insensitive EP during the reaction. The time course of EP hydrolysis could be quantitatively analyzed in terms of the following reaction mechanism. (formula; see text) The decomposition of EDTA-insensitive EP required Mg2+ outside the vesicles and was competitively inhibited by extravesicular Ca2+. The decomposition of EDTA-sensitive EP was inhibited by Ca2+ inside the vesicles but not by external Ca2+. The linear relationships between the inverse of the rate constants of EP decomposition during the initial phase and the intravesicular CaCl2 concentrations suggested that decomposition of EDTA-sensitive EP was inhibited by the binding of 1 mol of intravesicular Ca2+ to 1 mol of EP. Furthermore, Mg2+ inside the vesicles scarcely affected the inhibition of EP hydrolysis by intravesicular Ca2+. These results suggested that magnesium ions are not counter-transported during the active transport of calcium by SR vesicles.
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PMID:Inhibition of hydrolysis of phosphorylated Ca2+,Mg2+-ATPase of the sarcoplasmic reticulum by Ca2+ inside and outside the vesicles. 315 20


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