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)

Calmodulin and troponin C exhibit calcium-dependent binding of 1 mol/mol of dynorphin. The dissociation constants of the complexes, determined in 0.20 N KC1-1.0 mM CaCI2, pH 7.3, are 0.6 microM for calmodulin, 2.4 microM for rabbit fast skeletal muscle troponin C, and 9 microM for bovine heart troponin C. Experiments with deletion peptides of dynorphin show that peptide chain length and especially charge affect the binding of the peptides by calmodulin. Dynorphin, but not mastoparan or melittin, inhibits adenosinetriphosphatase activity in a reconstituted rabbit skeletal muscle actomyosin assay. The inhibition is partially reversed by the addition of calmodulin or troponin C in the presence of calcium. Calmodulin also exhibits calcium-dependent binding of a synthetic peptide corresponding to positions 104-115 of rabbit fast skeletal muscle troponin I. Mastoparan is a tetradecapeptide from the vespid wasp having exceptional affinity for calmodulin, with Kd approximately 0.3 nM [Malencik, D.A., & Anderson, S.R. (1983) Biochem. Biophys. Res. Commun. 114, 50]. The addition of 1 mol/mol of mastoparan to the complex of calmodulin with dynorphin results in complete dissociation of dynorphin. Similar titrations of the skeletal muscle troponin C-dynorphin complex produce a gradual dissociation consistent with a dissociation constant of 0.2 microM for the troponin C-mastoparan complex. Fluorescence anisotropy measurements using the intrinsic tryptophan fluorescence of mastoparan X show strongly calcium-dependent binding by proteolytic fragments of calmodulin. binding by proteolytic fragments of calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Peptide binding by calmodulin and its proteolytic fragments and by troponin C. 614

AR-L 115BS, a benzimidazole derivative, is a positive inotropic agent that has been shown to increase active tension development and unloaded shortening velocity of chemically skinned heart muscle preparations at submaximal activating levels of free Ca++. We measured the effect of AR-L on relations between free Ca++, bound Ca++ and ATPase activity of dog cardiac myofibrils. At pCa 6, 100-300 micrometers AR-L increased myofibrillar ATPase activity maximally by about 30%. The concentration of AR-L giving half-maximal activation of myofibrillar ATPase activity was about 10 micrometers, and is similar to plasma concentrations associated with elevated contractility in intact animals. There was no effect of AR-L on myofibrillar ATPase activity at pCa 5 or 8, and the relation between pCa and percent activation of myofibrillar ATPase activity was shifted to the left by 0.4-0.5 pCa units in the presence of 100 micrometers AR-L. Calcium binding by cardiac myofibrils was increased by AR-L in the presence and absence of MgATP by 0.2-0.3 nmol/mg myofibrillar protein over a broad range of free Ca++ concentrations, a result suggesting that AR-L increases the affinity of myofibrillar troponin C for Ca++. The shift in the pCa giving half maximal and myofibrillar ATPase activity induced by raising the free Mg++ from 1.0 to 10 mm was unaffected by AR-L. These results indicate that the positive actions of AR-L 115BS on cardiac contractility may involve direct activation of myofibrils by virtue of an increased affinity of thin filament receptors for Ca++.
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PMID:Stimulation of Ca++ binding and ATPase activity of dog cardiac myofibrils by AR-L 115BS, a novel cardiotonic agent. 621 30

1. Hybrid or reconstituted troponins were prepared from troponin components of rabbit skeletal muscle and porcine cardiac muscle and their effect on the actomyosin ATPase activity was measured at various concentrations of Ca2+ or Sr2+. The Ca2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing cardiac troponin I was slightly higher than that with troponin containing skeletal troponin I. The Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing skeletal troponin C was higher than that with troponin containing cardiac troponin C. 2. Reconstituted cardiac troponin was phosphorylated by cyclic AMP-dependent protein kinase. The Ca2+ sensitivity of actomyosin ATPase with cardiac troponin decreased upon phosphorylation of troponin I; maximum ATPase activity was depressed and the Ca2+ concentration at half-maximum activation increased. On the other hand, phosphorylation of troponin I did not change Sr2+ sensitivity. 3. The inhibitory effect of cardiac troponin I on the actomyosin ATPase activity was neutralized by increasing the amount of brain calmodulin at high Ca2+ and Sr2+ concentrations but not at low concentrations. 4. ATPase activity of actomyosin with a mixture of troponin I and calmodulin was assayed at various concentrations of Ca2+ or Sr2+. The Ca2+ or Sr2+ sensitivity of actomyosin ATPase containing skeletal troponin I was approximately the same as that of actomyosin ATPase containing cardiac troponin I. Phosphorylation of cardiac troponin I did not change the Ca2+ sensitivity of the ATPase. 5. The Ca2+ or Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin I-T-calmodulin was higher than that of actomyosin ATPase with the mixture of troponin I and calmodulin. Maximum ATPase activity was lower than that with the mixture of troponin I and calmodulin.
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PMID:Sensitivity of actomyosin ATPase to calcium and strontium ions. Effect of hybrid troponins. 622 22

Equilibrium-binding studies at 4 degrees C show that, in the instance of crayfish, troponin C contains only one Ca-binding site with an affinity in the range of physiological free [CA2+] (K = 2 X 10(5) M-1). At physiological levels of Mg2+, this site does not bind Mg2+. In the complexes of troponin C-troponin I, troponin and troponin-tropomyosin, the regulatory Ca-specific site exhibits a 10- to 20-fold higher affinity (K = 2-4 X 10(6) M-1). The latter affinity is reduced to that of troponin C upon incorporation of the troponin-tropomyosin complex into the actin filament (regulated actin), as determined at 4 degrees C by the double isotope technique. The Ca-binding constant is again shifted to a higher value (7 X 10(6) M-1) when regulated actin is associated with nucleotide-free myosin. Both crayfish myofibrils and rabbit actomyosin regulated by crayfish troponin-tropomyosin display a steep rise in ATPase activity with [Ca2+]. Comparison of the pCa/ATPase relationship and the Ca-binding properties at 25 degrees C for the crayfish troponin-regulated actomyosin indicates that while the threshold [Ca2+] for activation corresponds to the range of [Ca2+] where the regulatory site in its low affinity state (K = 1 X 10(5) M-1) starts to bind Ca2+ significantly, full activation is reached at [Ca2+] for which the Ca-specific site in its high affinity state (K = 3 X 10(6) M-1) approaches saturation. These results suggest that, in the actomyosin ATPase cycle, there are at least two calcium-activated states of regulated actin (one low and one high), the high affinity state being induced by interactions of myosin with actin in the cycle.
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PMID:Regulation of actomyosin ATPase by a single calcium-binding site on troponin C from crayfish. 623 21

The aim of experiments described here was to test whether deactivation of cardiac myofibrils in acidic pH is associated with decreases in amounts of calcium bound to myofilament troponin. We determined the amounts of myofibrillar bound calcium attributable to troponin, from measurements of calcium binding to myofibrils and to myosin and from determination of the troponin C content of the myofibrillar preparations (0.40 nmol troponin C/mg protein). In measurements done at 2 mM free magnesium, 2 mM (magnesium-adenosine triphosphate, ionic strength 0.12, 22 degrees C, the pCa50 (-log of the half maximally activating molar free calcium) for myofibrillar magnesium-adenosine triphosphatase activity was 5.87 at pH 7.0, 5.49 at pH 6.5, and 5.04 at pH 6.2. This change in calcium sensitivity of myofibrillar magnesium-adenosine triphosphatase activity was present whether or not ethyleneglycol-bis(beta-aminoethyl ether)-N, N'-tetraacetic acid, was used to buffer the free calcium and whether or not myofibrillar troponin I had been phosphorylated by cyclic adenosine 3',5'-monophosphate-dependent protein kinase. However, the change in pCa50 of myofibrillar adenosine triphosphatase activity induced by acidic pH, was greater when free magnesium was reduced from 2.0 to 0.05 mM, and less when free magnesium was increased from 2.0 mM to 10 and 15 mM. The change in pCa50 with acidic pH was less if the ionic strength was reduced from 0.12 to 0.035 M. The magnesium-adenosine triphosphatase activity of troponin/tropomyosin-free myofibrils was independent of pCa and unaffected by a reduction of pH from 7.0 to 6.5. The affinity of myofibrillar troponin C for calcium decreased as pH was reduced from 7.0 to 6.5 and to 6.2 with and without ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid, and in a manner predicted from the effect of acidic pH on pCa50 for myofibrillar activation. Our results are consistent with the idea that at least part of the mechanism responsible for deactivation of the adenosine triphosphatase activity of cardiac myofilaments in acidic pH is a reduction in the affinity of myofibrillar troponin C for calcium.
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PMID:Inhibition of the activation and troponin calcium binding of dog cardiac myofibrils by acidic pH. 623 79

Localization and quantification studies were carried out on bay-scallop (Aequipecten irradians) striated-muscle troponin C- and troponin I-like proteins. Indirect immunofluorescence microscopy of scallop myofibrils stained with either rabbit anti-(scallop troponin I) or anti-(scallop troponin C) antibodies shows staining of all I-bands observed. The results of quantification studies using sodium dodecyl sulfate poly-acrylamide-gel electrophoresis of untreated scallop myofibrils, washed scallop myofibrils, and isolated scallop thin filaments indicate an actin/tropomyosin/troponin-C molar rationn of 7:1:1. The molar ratio for troponin I could not be determined in untreated myofibrils because of interfering bands; in washed myofibrils a value of 0.6 mol of troponin I/mol of tropomyosin was found. Purified scallop troponin C binds Ca2+ and interacts with scallop troponin I to relieve troponin I-induced inhibition of actomyosin ATPase. Although scallop troponin C is an acidic protein, it appears to be less acidic than troponin C from higher organisms. A calmodulin-like protein has been isolated from scallop striated muscle that activates bovine brain phosphodiesterase to the same extent as does brain calmodulin. Its amino acid composition and its electrophoretic mobility on alkaline 6 M-urea/polyacrylamide gels differs from that of scallop troponin C, and it appears not to be associated with thin filaments.
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PMID:The stoichiometry and location of troponin I- and troponin C-like proteins in the myofibril of the bay scallop, Aequipecten irradians. 624 69

1. Porcine cardiac native tropomyosin was phosphorylated by bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. Most of the phosphate incorporation was observed in troponin I, the maximum of which was 0.7 mol of Pi per mol of troponin I. 2. In the presence of phosphorylated native tropomyosin, actomyosin ATPase activity was 15-40% lower than that in the presence of the unphosphorylated preparation at all calcium ion concentrations (1.5 x 10(-8) M-2.4 x 10(-5) M). Half-maximum activation of ATPase was obtained with a concentration of 7 x 10(-7) M Ca2+ (unphosphorylated) and 1.3 x 10(-6) M Ca2+ (phosphorylated), respectively. Maximum ATPase activity was reached with 3 x 10(-6) M Ca2+ (unphosphorylated) and 1.0 x 10(-5) M Ca2+ (phosphorylated). 3. Porcine cardiac troponin I isolated by affinity chromatography inhibited ATPase activity of desensitized actomyosin in the presence of tropomyosin. There was little difference between phosphorylated troponin I and a control preparation with regard to the inhibitory effect of ATPase activity. 4. Troponin C from rabbit skeletal muscle neutralized the inhibitory effect of troponin I. The minimum amount of troponin C required for complete neutralization was approximately equimolar to troponin I. The inhibitory effect of phosphorylated troponin I was neutralized by troponin C less effectively than that of unphosphorylated preparation.
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PMID:Effect of phosphorylation of porcine cardiac troponin I by 3':5'-cyclic AMP-dependent protein kinase on the actomyosin ATPase activity. 628 30

Adrenergic stimulation alters functional dynamics of the heart by mechanisms most likely involving cyclic AMP (cAMP)-dependent protein phosphorylation. In vitro studies indicate that the myofibrils and sarcoplasmic reticulum (SR) may act as effectors of the adrenergic stimulation. cAMP-dependent phosphorylation of troponin I (TnI), one of the regulatory proteins of cardiac myofibrils, results in a decreased steady-state affinity of troponin C (TnC) for calcium, an increase in the off-rate for Ca2+ exchange with TnC, and a rightward shift of the relation between free Ca2+ and myofibrillar force or ATPase. Phosphorylation of phospholamban, a regulatory protein of cardiac SR, results in an increased velocity of Ca2+ transport by SR vesicles, an increased affinity of the transport protein for Ca2+, and an increased turnover of elementary steps of the ATPase reaction. These in vitro findings support the hypothesis that the inotropic response of the heart to catecholamine stimulation involves phosphorylation of TnI and phospholamban. Our in vivo studies with perfused rabbit hearts show that during the peak of the inotropic response to isoproterenol there is a simultaneous phosphorylation of TnI and an 11,000-dalton protein in the SR, most likely the monomeric form of phospholamban.
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PMID:Coordination of cardiac sarcoplasmic reticulum and myofibrillar function by protein phosphorylation. 629 80

It was shown that 3,3'-dipropylthiocarbocyanine iodide diS-C3-(5) can be used as a fluorescent probe for registration of conformational changes in calmodulin and troponin C, as well as for determination of concentrations of these Ca-binding proteins in experimental samples. The sensitivity of the method (10(-7) M) is only 5 times less than that of determination of calmodulin by phosphodiesterase and by 1 or 2 orders of magnitude more than that of other techniques based on conformational changes of the protein. The spectral parameters of the fluorescent probe allow to conduct the measurements in turbid media and in the presence of many other optically active substances. Evidence is given testifying the promiscuity of the use of this approach to the study of conformational changes in calmodulin under the action of Ca2+, Mg2+, monovalent cations, temperature and target proteins (troponin I, phosphodiesterase). using phosphodiesterase and Ca-ATPase, it was shown that under certain conditions diS-C3-(5) as well as other amphypathic compounds can modify the activity of calmodulin-dependent enzymes.
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PMID:[Use of 3,3'-dipropylthiodicarbocyanine iodide diS-C3-(5) for the study of conformational changes and detection of Ca-binding proteins]. 632 70

Myosin and actin competition tests indicated the presence of both thin-filament and myosin-linked Ca2+-regulatory systems in pig aorta and turkey gizzard smooth-muscle actomyosin. A thin-filament preparation was obtained from pig aortas. The thin filaments had no significant ATPase activity [1.1 +/- 2.6 nmol/mg per min (mean +/- S.D.)], but they activated skeletal-muscle myosin ATPase up to 25-fold [500 nmol/mg of myosin per min (mean +/- S.D.)] in the presence of 10(-4) M free Ca2+. At 10(-8) M-Ca2+ the thin filaments activated myosin ATPase activity only one-third as much. Thin-filament activation of myosin ATPase activity increased markedly in the range 10(-6)-10(-5) M-Ca2+ and was half maximal at 2.7 x 10(-6) M (pCa2+ 5.6). The skeletal myosin-aorta-thin-filament mixture gave a biphasic ATPase-rate-versus-ATP-concentration curve at 10(-8) M-Ca2+ similar to the curve obtained with skeletal-muscle thin filaments. Thin filaments bound up to 9.5 mumol of Ca2+/g in the presence of MgATP2-. In the range 0.06-27 microM-Ca2+ binding was hyperbolic with an estimated binding constant of (0.56 +/- 0.07) x 10(6) M-1 (mean +/- S.D.) and maximum binding of 8.0 +/- 0.8 mumol/g (mean +/- S.D.). Significantly less Ca2+ bound in the absence of ATP. The thin filaments contained actin, tropomyosin and several other unidentified proteins. 6 M-Urea/polyacrylamide-gel electrophoresis at pH 8.3 showed proteins that behaved like troponin I and troponin C. This was confirmed by forming interspecific complexes between radioactive skeletal-muscle troponin I and troponin C and the aorta thin-filament proteins. The thin filaments contained at least 1.4 mumol of a troponin C-like protein/g and at least 1.1 mumol of a troponin I-like protein/g.
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PMID:Calcium ion-regulated thin filaments from vascular smooth muscle. 644 98


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