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:3.6.4.1 (myosin ATPase)
1,140 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of crosslinking of F-actin with glutaraldehyde on the contractility of muscle ghost fiber containing reconstituted thin filament (i.e. F-actin-tropomyosin-troponin complex) and irrigated with myosin was investigated. The results show that: (i) crosslinking inhibited development of isometric tension and shortening of the fiber in the presence of MgATP, (ii) superprecipitation of the complex of myosin with crosslinked thin filament was considerably delayed, (iii) crosslinking inhibited neither binding of myosin heads to the filament nor activation of myosin ATPase. It is suggested that alterations of actin structure due to the formation of intra- and/or intermonomer crosslinks can essentially affect the process of contractility.
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PMID:The effect of crosslinking of thin filament with glutaraldehyde on the contractility of muscle fiber. 621

Initial studies on molluscan muscle regulation indicated that thin filaments do not confer Ca2+-dependence on vertebrate myosin ATPase, and hence that molluscan muscles do not possess thin filament-linked regulatory systems. Subsequently it was shown that molluscan thin filaments do, in fact, impart Ca2+-sensitivity but only at Mg2+ concentrations greater than those used in the earlier studies. In the present study it is shown that Mg2+ prevents significant dissociation of tropomyosin and troponin subunits from thin filaments at the low monovalent ion concentrations typically employed to assay actomyosin ATPase; as a result Mg2+ allows expression of the molluscan thin filament regulatory system under these conditions.
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PMID:The ionic requirements for regulation by molluscan thin filaments. 622 62

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

The two light chains of Physarum myosin have been purified in a 1:1 ratio with a yield of 0.5-1 mg/100 g of plasmodium and a purity of 40-70%; the major contaminant is a 42,000-dalton protein. The 17,700 Mr Physarum myosin light chain (PhLC1) binds to scallop myofibrils, providing the regulatory light chains (ScRLC) have been removed. The 16,500 Mr light (PhLC2) does not bind to scallop myofibrils. The calcium control of scallop myosin ATPase is lost by the removal of one of the two ScRLC's and restored equally well by the binding of either PhLC1 or rabbit skeletal myosin light chains. When both ScRLC's are removed, replacement by two plasmodial light chains does not restore calcium control as platelet or scallop light chains do. Purified plasmodial actomyosin does not bind calcium in 10(-6) M free calcium, 1 mM MgCl2. No tropomyosin was isolated from Physarum by standard methods. Because the Physarum myosin light chains can substitute only partially for light chains from myosin linked systems, because calcium does not bind to the actomyosin, and because tropomyosin is apparently absent, the regulation of plasmodial actomyosin by micromolar Ca++ may involve other mechanisms, possibly phosphorylation.
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PMID:Hybrids of Physarum myosin light chains and desensitized scallop myofibrils. 645 52

Regulated actin, F-actin plus troponin-tropomyosin, activates the myosin ATPase in the presence but not in the absence of calcium. Ultracentrifugation has been used to examine the interaction of regulated actin with two proteolytic fragments of myosin, heavy meromyosin (HMM), a two-headed species, and subfragment 1 (S-1), a single head. In the presence of ATP, the association constant (Ka) for the binding of S-1 to regulated actin is approximately 1.5 X 10(4) M-1, whether or not calcium is present. This is true for S-1 with and without the Mr = 19,000 phosphorylatable light chain. These results confirm the observations of Chalovich et al. (Chalovich, J., Chock, P. B., and Eisenberg, E. (1981) J. Biol. Chem. 256, 575-578) and support their suggestion that the inhibition of the regulated actin-activated ATPase of S-1 by removal of calcium does not result from preventing S-1 binding. The binding of HMM to regulated actin in the presence of ATP, however, is calcium sensitive. Removal of calcium results in approximately a 4-fold decrease in Ka, from 1.1 X 10(4) M-1 to 2.5 X 10(3) M-1. The results with HMM are more easily reconciled with the low stiffness of relaxed muscle and suggest a functional difference between S-1 and HMM.
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PMID:Calcium-sensitive binding of heavy meromyosin to regulated actin in the presence of ATP. 645 6

Actin-myosin subfragment-1 (SF-1) or actin-heavy meromyosin is dissociated by the binding of ADP and vanadate (Vi) under conditions such that ADP alone does not dissociate the complex. The association constant of the stable complex M.ADP.Vi, in which M indicates myosin [Goodno, C. C. (1979) Proc. Natl. Acad. Sci. USA 76, 2620-2624] with actin is smaller than the average association constant of the intermediate states of the actin-SF-1 ATPase cycle. Actin-SF-1 ATPase activity is 90% inhibited by ADP plus vanadate. The reaction of actin with M.ADP.Vi produces a slow release of ADP and vanadate and quantitative recovery of ATPase activity. The rate of dissociation of ligands was almost linear in actin concentration; consequently, the rate constant of dissociation could only be roughly estimated as 0.5-1 sec-1. The rate of dissociation of ADP and vanadate is thus increased by a factor of 10(5) compared to M.ADP.Vi. The rate of release of ligands by regulated actin (actin-tropomyosin-troponin) was reduced to 1/10th to 1/20th by removal of calcium ion. Therefore the M.ADP.Vi complex has the properties of a more stable analogue of the myosin-ADP-phosphate complex that is generated in the normal ATPase cycle. The activation of ligand release (ratio of rate of dissociation of ADP and vanadate from actomyosin relative to myosin) is much larger than the activation of myosin ATPase by actin, whereas the actual rates of the reactions are much slower.
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PMID:Inhibition of actomyosin ATPase by vanadate. 645 80

Cofilin, a 21 000 molecular weight protein of porcine brain, reacts stoichiometrically with actin in a 1:1 molar ratio. Upon binding of cofilin, the fluorescence of pyrene-labeled actin under polymerizing conditions is changed into the monomer form, irrespective of whether cofilin is added to actin before or after polymerization. Cofilin decreases the viscosity of actin filaments but increases the light-scattering intensity of the filaments. The centrifugation assay and the DNase I inhibition assay demonstrate that cofilin binds to actin filaments in a 1:1 molar ratio of cofilin to actin monomer in the filament and that cofilin increases the monomeric actin to a limited extent (up to 1.1-1.5 microM monomer) in the presence of physiological concentrations of Mg2+ and KCl. Cofilin is also able to bind to monomeric actin, as demonstrated by gel filtration. Electron microscopy showed that actin filaments are shortened and slightly thickened in the presence of cofilin. No bundle formation was observed in the presence of various concentrations of cofilin. The gel point assay using an actin cross-linking protein and the nucleation assay also suggested that cofilin shortens the actin filaments and hence increases the filament number. Cofilin blocks the binding of tropomyosin to actin filaments. Tropomyosin is dissociated from actin filaments by the binding of cofilin to actin filaments. Cofilin was found to inhibit the superprecipitation of actin-myosin mixtures as well as the actin-activated myosin ATPase. All these results suggest that cofilin is a new type of actin-associated protein.
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PMID:Cofilin, a protein in porcine brain that binds to actin filaments and inhibits their interactions with myosin and tropomyosin. 650 22

The Ca2+-binding component of troponin (TnC) and its proteolytic fragments containing Ca2+-binding sites I-III (TH1) or sites III and IV (TR2C) have been labeled with the fluorescent probes dansylaziridine (DANZ) at methionine 25 or 5-(iodoacetamidoethyl)amino-naphthalene-1-sulfonic acid (AEDANS) at cysteine-98. These probes report binding of Ca2+ to the low and high affinity sites, respectively. Fluorescence changes as a function of [Ca2+] were measured for the free peptides, their complexes with troponin I + troponin T, and these complexes bound to actin-tropomyosin in the presence of Mg2+ and ATP with and without myosin. An apparent Hill coefficient of 1.0-1.1 has been obtained for the Ca2+-induced fluorescence changes in TnC, its fragments, and their ternary complexes regardless of the label used. When a ternary complex containing appropriately labeled TnC or its fragment is bound to the actin-tropomyosin complex, the Hill coefficient for the titration of the low affinity sites increases to 1.5-1.6 and further increases to greater than 2 in the presence of myosin. To interpret the apparent Hill coefficients, we used a model containing two binding sites and a single reporter of the conformational change. Hill coefficients between 1.0 and 1.2 can be obtained for the fluorescence change without true cooperativity in metal binding, depending on the mechanism of the fluorescence change; i.e. the contribution of the singly or doubly occupied species to the fluorescence change. A Hill coefficient between 1.2 and 2, however, always indicates cooperativity in binding independently of the mechanism. Thus, our finding that fluorescence titrations of Ca2+ binding to TnCDANZ bound to actin-tropomyosin exhibit a Hill coefficient of 1.5 in the absence of myosin and 2.4 in its presence indicates the existence of true positive cooperativity in metal binding to sites I and II. No cooperativity was observed for AEDANS-labeled complexes that reflect Ca2+-binding to the high affinity sites. Plots of the Ca2+ dependence of myosin ATPase activity activated by actin-tropomyosin in the presence of any of the troponin complexes used had apparent Hill coefficients of approximately 4. The higher value suggests cooperative interactions in the activation of ATPase beyond those involved in Ca2+-binding to the Ca2+-specific sites.
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PMID:Cooperative binding to the Ca2+-specific sites of troponin C in regulated actin and actomyosin. 664 69

Lysine 372 of N-ethylmaleimide actin was specifically (60%) labeled by 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole chloride (NBD-Cl), which also reacted with lysines on cyanogen bromide fragment 17 (20%) and other undetermined residues (20%). Isolation of N-ethylmaleimide peptides and two-dimensional peptide mapping demonstrated that 90% of bound N-ethylmaleimide was attached to an adjacent residue, cysteine 373, independent of the polymerization state of actin during the labeling reaction. Formation of NBD cysteine severely inhibited lysine modification. After N-ethylmaleimide blockage of cysteine 373, lysine labeling with NBD was greatly accelerated. The kinetics of formation of fluorescent compounds were biphasic, with fluorescence decreasing upon prolonged incubation of actin in NBD-Cl. Lysine 372 of purified NBD actin reproducibly responded to polymerization by a 2.2- to 2.3-fold enhancement of fluorescence. By contrast, interaction of NBD actin with several actin-binding proteins caused only very small or undetectable changes in fluorescence intensity: 10% enhancement on myosin subfragment 1 binding, about 6% quenching by DNase I, and no change at all by tropomyosin-troponin. Despite its sensitivity to polymerization the probe did not affect it. Native and modified actin polymerized randomly indicating that the rate constants for polymerization remained the same. Labeling actin with NBD did not diminish its cofactor activity for myosin ATPase activity. Contrary to previous reports we observed that myosin subfragment 1 (single myosin heads) caused actin polymerization in the absence of salt.
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PMID:7-Chloro-4-nitrobenzeno-2-oxa-1,3-diazole actin as a probe for actin polymerization. 700 20

It is shown that smooth muscle actin preparations, produced with the help of techniques described in literature, have contaminations of tropomyosin and proteins with the molecular weight of 250 000, 80 000 and 18 000. The composition of contaminations depends on the method of preparation and conditions of dissociation of native actomyosin material. A method is proposed for actin separation from acetone-treated actomyosin followed by salting out with 50 mM MgCl2. Gel-electrophoresis in the presence of Na-dodecylsulphate has shown that the actin preparations are homogeneous and have the same cofactor activity for the myosin ATPase as actin from the rabbit skeletal muscle. It is supposed that in the cytoplasm of smooth muscle cells as well as in the non-muscle ones there is a considerable part of actin is the monomer form. It gets easily lost in the process of fibrillar actin separation. This may account for low actin output in spite of its relatively high contents in smooth muscles.
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PMID:[Features of the preparation and estimation of homogeneity and native state of smooth muscle actin]. 713 7


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