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 effects of bivalent (Mg2+, Ca2+, Sr2+) and monovalent (K+, Na+, NH4+) cations on the ATPase activity of subfragment 1 of myosin (SI) with a decreased Mg2+ content (EDTA-SI) were studied. Mg2+ activate the EDTA-SI ATPase, but only in the absence of other activating cations. K+, NH4+, a2+ and Sr2+ have a much stronger activating effect on EDTA-SI ATPase than on Mg-SI (SI enriched with Mg2+) ATPase. Monovalent cations inhibit Mg2+-ATPase and Ca2+-ATPase of EDTA-SI, while K+ and NH4+ activate Sr2+-ATPase of EDTA-SI. Based on experimental results and literary data, a hypothesis on the participation of the cations in the functioning of myosin ATPase was postulated. This hypothesis entails the existence of two closely interconnected cation-binding sites in the vicinity of the myosin active center (one for bivalent and one for monovalent cations); the ATPase activity of myosin is at any moment dependent on the nature of cations present in these two sites. An attempt to explain the role of the cations in the accomplishment of the ATPase reaction by myosin was made.
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PMID:[Role of bivalent and monovalent cations in the functioning of myosin ATPase]. 645 45

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

The reactivity of thiol groups in cardiac myosin obtained from porcine ventricles was compared with that in rabbit skeletal myosin. 1. The specific thiol group, S2, of cardiac myosin became reactive to N-ethylmaleimide (NEM) upon the addition of Mg2+-ATP or Mg2+-ADP, as in the case of skeletal myosin, although to a lesser degree. Enhancement of the reactivity of S2 resulting from cooperative interaction between F-actin and Mg2+-ATP, but not Mg2+-ADP, which was first found in skeletal myosin, was also observed in cardiac myosin. 2. The total number of 5,5'-dithiobis(2-nitrobenzoate) (DTNB)-reactive thiols decreased in the presence of Mg2+ADP and decreased further in the presence of Mg2+-ATP for both myosins, in contrast to the concomitant increase in the reactivity of S2. The decrement was smaller in cardiac myosin than in skeletal myosin. 3. The DTNB-reactive thiols were classified into three groups with respect to their reactivity. The third or most slowly reacting thiol group, was found to be remarkably susceptible to Ca2+ at physiological concentrations (10(-8)-10(-6) M) in the presence of Mg2+-ADP for cardiac myosin, but in striking contrast, in the presence and relatively long-lived intermediate of myosin ATPase, the structure of which is sensitive to Ca2+, may be different in cardiac and skeletal myosins.
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PMID:Actin-induced local conformational change in the myosin molecule. III. Reactivity of S2 thiol and DTNB-reactive thiols of porcine cardiac myosin. 689 37

It was shown that binding of the cation Eu3+ to myosin subfragment I (SI) results in fluorescence with a maximum at 595 nm which is increased as EuCl3 concentration rises. The ATPase activity of SI is simultaneously enhanced. An addition of bivalent cations (Ca2+ and Mg2+) causes quenching of fluorescence of the bound Eu3+ by 8-12%, which corresponds to Eu3+-ATPase inhibition by Mg2+. At low (down to 0.1 mM) concentrations of Eu3+ no fluorescence quenching by Mg2+ or Ca2+ takes place; under these conditions Mg2+ activate ATPase of SI in the presence of Eu3+. Eu3+ activate SI ATPase in the presence of low (down to 0.1 mM) concentrations of Ca2+, but exerts an inhibition action at high concentrations of Ca2+. NaCl does not affect the fluorescence intensity of bound Eu3+ but considerably inhibits the ATPase activity of SI in the presence of EuCl3. An existence of a bivalent cation binding site in the vicinity if the SI active center is postulated. Eu3+ whose ionic radius is close to that of Ca2+ interacts with protein surface and occupies this site as well, thus determining the activation of SI ATPase and can be replaced from it by Ca2+ and Mg2+, but not by Na+. Hence bivalent and monovalent cations are bound at different sites. The data obtained provide another proof in favour of a hypothesis suggesting that the regulation of myosin ATPase activity by bivalent and monovalent cations can be mediated by binding of these cations to the protein.
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PMID:[Study of cation binding to myosin subfragment I using the fluorescent probe Eu3+]. 713 65

Calcium binding proteins mediate a large number of cellular processes. These processes respond to micromolar fluctuations of cytosolic calcium in the presence of a large excess of magnesium. The metal binding sites present in these proteins are either calcium-specific (regulatory sites) or capable of binding both calcium and magnesium (structural sites). Using site-directed mutagenesis we were able to convert the single Ca2+/Mg2+ site present in chicken smooth muscle myosin regulatory light chain (RLC) into a Ca(2+)-specific site. The replacement of the aspartic acid present in the 12th position (-Z coordinating position) of the metal binding loop with a glutamic acid increases calcium affinity and abolishes magnesium binding, rendering the site calcium-specific. To explain this observation, we hypothesize that restrictions on the ability of side chains to change conformation, contributing one (for Mg2+ binding) or two (for Ca2+ binding) coordinations could alter the metal specificity in EF-hands. Other mutations which decrease or abolish calcium binding have also been characterized. When used to substitute the endogenous scallop myosin RLC, these mutants were capable of restoring the Ca2+ regulation to the actin-activated myosin ATPase demonstrating that in these hybrid myosins, the regulatory function of the Ca(2+)-specific site (present on the essential light chain) does not depend on the occupancy of the Ca2+/Mg2+ site (present on the regulatory light chain).
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PMID:Determinants of ion specificity on EF-hands sites. Conversion of the Ca2+/Mg2+ site of smooth muscle myosin regulatory light chain into a Ca(2+)-specific site. 789 23

An ATPase activity is associated with maize (Zea mays) annexins. It has a pH optimum of 6.0, shows Michaelis-Menten kinetics and is not stimulated by Ca2+, Mg2+, EDTA or KCl; it is not inhibited by vanadate, molybdate, nitrate or azide, but N-ethylmaleimide inhibits by approximately 30% at 1-2 mM. These properties indicate that the activity is unlike other ATPases, although it has many features in common with the myosin ATPase. Gel filtration shows that the ATPase activity is mainly associated with a 68 kDa protein that is extracted with the p33/p35 annexins and cross-reacts with antibodies to these proteins.
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PMID:Identification and characterization of ATPase activity associated with maize (Zea mays) annexins. 798 Apr 36

Myocardial ischemia is characterized by a decrease in phosphocreatine (PCr) and Mg(2+)-ATP contents as well as an accumulation of myosin ATPase reaction products (inorganic phosphate [P(i)], protons, and Mg(2+)-ADP). The possibility that these metabolites play a role in rigor tension development was checked in rat ventricular Triton X-100-skinned fibers. Rigor tension was induced by stepwise decreasing [Mg(2+)-ATP] in the presence or in the absence of 12 mmol/L PCr. To mimic the diastolic ionic environment of the myofibrils, [free Ca2+] was set at 100 nmol/L (pCa 7); [free Mg2+], at 1 mmol/L; and ionic strength, at 160 mmol/L. In control conditions (pH 7.1, with no added P(i) or Mg(2+)-ADP), the pMg(2+)-ATP for half-maximal rigor tension (pMg(2+)-ATP50) was 5.07 +/- 0.03 in the presence of PCr. After withdrawal of PCr, the pMg2+)-ATP50 value was shifted toward higher Mg(2+)-ATP values (3.57 +/- 0.03). Addition of 20 mmol/L P(i) shifted the pMg(2+)-ATP50 to 3.71 +/- 0.04 (P < .05) in the absence of PCr and in the opposite direction to 4.98 +/- 0.02 (P < .01) in the presence of PCr. Acidic pH (6.6) strongly increased pMg(2+)-ATP50 in both the absence (3.90 +/- 0.03, P < .001) and presence (5.44 +/- 0.02, P < .001) of PCr. Conversely, Mg(2+)-ADP (250 mumol/L) decreased pMg(2+)-ATP50 to 3.26 +/- 0.06 (P < .001) in the absence of PCr; at pMg(2+)-ATP 4, no rigor tension was observed until PCr concentration was decreased to < 2 mmol/L. At acidic pH, maximal rigor tension was lower by 29% compared with control conditions, whereas in the presence of Mg(2+)-ADP, maximal rigor tension developed to 143% of the control value; P(i) had no effect. The tension-to-stiffness (measured by the quick length-change technique) ratio was lower in rigor (no PCr and pMg(2+)-ATP 6) than during Ca2+ activation in the presence of both PCr and ATP. Compared with control rigor conditions, this parameter was unchanged by Mg(2+)-ADP and decreased by acidic pH, suggesting a proton-induced decrease in the amount of force per crossbridge. In addition to their known effects on active tension, Mg(2+)-ADP and protons affect rigor tension and influence ischemic contracture development. It is concluded that ischemic contracture and increased myocardial stiffness may be mediated by a decreased PCr and local Mg(2+)-ADP accumulation. This emphasizes the importance of myofibrillar creatine kinase activity in preventing ischemic contracture.
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PMID:Myocardial ischemic contracture. Metabolites affect rigor tension development and stiffness. 815 39

We have previously shown that in human or pig whole erythrocytes, only a single 71-kDa polypeptide cross-reacts with the affinity-purified antibody to pig platelet caldesmon (der Terrossian et al., 1989). In the present paper, we demonstrate that this polypeptide represents a genuine caldesmon which remains attached to the membrane prepared in the presence of an excess of free Mg2+ but not in its absence. Immunoreactivity of this peptide is specific towards the antibody to pig platelet caldesmon since it is not labelled with antibodies to other components of the red cell membrane. Erythrocyte caldesmon was purified to 95% homogeneity and displays well known characteristics of caldesmons from other sources. Together with tropomyosin, it has the ability to regulate platelet actin-activated rabbit skeletal muscle myosin ATPase activity. The stoichiometry of 1 caldesmon/1 tropomyosin/7-9 actin molecules indicates that the amount of caldesmon, in the red cell membrane, corresponds precisely to the amount of tropomyosin. Immunofluorescent labelling of whole erythrocytes gave similar punctate patterns with purified antibodies to myosin, to caldesmon, to tropomyosin and to actin (but not to spectrin), suggesting colocalization of these proteins. Together, and for the first time, our results give strong evidence that caldesmon, bound on the actin protofilament, might represent the inhibitory component, so far uncharacterized, of a thin-filament-like system in erythrocyte.
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PMID:Purification and characterization of erythrocyte caldesmon. Hypothesis for an actin-linked regulation of a contractile activity in the red blood cell membrane. 830 18

The synthesis of [2-3H]ATP with specific activity high enough to use for 3H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single 3H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes. The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg2+, S1 converts [2-3H]ATP to [2-3H]ADP and the complex S1.Mg[2-3H]ADP has ADP bound in the active site. At 0 degrees C, 1D 3H NMR spectra of S1.Mg[2-3H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-3H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-3H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 degrees C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (Vi) to produce S1.Mg[2-2H]ADP.Vi shifts both peaks slightly more upfield without changing their widths or relative areas.
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PMID:[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1. 835 34


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