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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)

The reactivity of the sulfhydryl groups in myosin B to N-ethylmaleimide (NEM) was investigated under various conditions. Under the conditions where actin and myosin associate, i.e. at low ionic strength, only Mg2+-ATPase [EC 3.6.1.3] activity was markedly activated by NEM treatment, whereas coupling of EDTA-ATPase inhibition with Ca2+-ATPase activation, which was seen on blocking S1 of myosin A with NEM, was observed under conditions at which the dissociation of actomyosin occurs, i.e. at high ionic strength, suggesting the covering with actin of the S1 region of myosin. Nevertheless, APT accelerated the reactivity of S1 and S2 much more in the myosin B system than in myosin alone. NEM-modified myosin B ATPase exhibited a shift of the KCL dependence curve to high concentration, a shift of the maximum activation of ATPase activity to high Mg ion concentration and a suppression of substrate inhibition at high substrate concentrations. These all indicate that the blocking by NEM of Sa, the sulfhydryl group related to the activation of Mg2+-ATPase of myosin B, brings about an increase in the association of myosin and actin in the myosin B system, resulting in an activation of Mg2+-ATPase activity. In addition, the relationship between Sa and a sulfhydryl group(s) essential for Ca2+ sensitivity was discussed.
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PMID:The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. I. Relantionship of the sulfhydryl group responsible for Mg2+-ATPase activation to the S1 and S2 groups. 12 48

The binding of ADP to subfragment-1 was investigated by the gel filtration method. The amount of bound ADP was determined as a function of free ADP concentration. Linear Scatchard plots were obtained. The maximum binding number, 0.55 mole of ADP per 10(5) g of protein, and the dissociation constant, 1.6 x 10(-6) M, were obtained, using subfragment-1 prepared by tryptic digestion, in the presence of 0.083 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 25 degrees. Similar maximum numbers, about 0.5 mole per 10(5) g of protein, were obtained with subfragment-1 prepared by chymotryptic digestion of myosin or papain digestion of myofibrils. The maximum number did not depend on the KCl concentration or the temperature, while the dissociation constant decreased on decreasing either the KCl concentration or the temperature. Adenylyl imidodiphosphate binding to subfragment-1 prepared by chymotryptic digestion was also measured by the gel filtration method. The maximum binding number, 0.41 mole per 10(5) g of subfragment-1, and the dissociation constant, less than 10(-7) M, were obtained in the presence of 0.7 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 8 degrees. The difference absorbance at 288 nm of the difference absorption spectrum induced by ADP of subfragment-1 prepared by tryptic digestion was proportional to the amount of bound ADP. The steady-state ATPase rate of subfragment-1 prepared by tryptic digestion was inhibited competitively by ADP in the presence of MgCl2. The extent of the initial burst of ATPase [EC 3.6.1.3] decreased from 0.46 +/- 0.06 to 0.30 +/- 0.09 mole of Pi per 10(5) g of subfragment-1 on adding ADP to a level of 0.6 mM. Subfragment-1 prepared by tryptic digestion bound F-actin with a mole ratio of 1/0.96 of actin monomer. The binding was depressed by the addition of ADP. On the basis of these results, subfragment-1 preparations were assumed to be a half-and-half mixture of two kinds of protein, and properties of each protein are discussed.
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PMID:A study of the binding of adenosine diphosphate to myosin subfragment-1. 12 50

The control systems regulating muscle contraction in approximately 100 organisms have been categorized. Both myosin control and actin control operate simultaneously in the majority of invertebrates tested. These include insects, chelicerates, most crustaceans, annelids, priapulids, nematodes, and some sipunculids. Single myosin control is present in the muscles of molluscs, brachiopods, echinoderms, echiuroids, and nemertine worms. Single actin control was found in the fast muscles of decapods, in mysidacea, in a single sipunculid species, and in vertebrate striated muscles. Classification is based on functional tests that include measurements of the calcium dependence of the actomyosin ATPase activity in the presence and the absence of purified rabbit actin and myosin. In addition, isolated thin filaments and myosins were also analyzed. Molluscs lack actin control since troponin is not present in sufficient quantities. Even though the functional tests indicate the complete lack of myosin control in vertebrate striated muscle, it is difficult to exclude unambiguously the in vivo existence of this regulation. Both control systems have been found in animals from phyla which evolved early. We cannot ascribe any simple correlation between ATPase activity, muscle structure, and regulatory mechanisms.
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PMID:Regulation of muscular contraction. Distribution of actin control and myosin control in the animal kingdom. 12 78

The ATPase activity of myosin and contraction time in extensor digitorum longus muscle, soleus muscle and cardiac muscle was compared in mammals differing in size. It was shown that the myosin ATPase activity of homologous muscles decreases and contraction time increases with increasing size of animals. The rate of tryptic digestion of myosin, the electrophoretic pattern of light chains of myosin and the effect of p-chloromercuribenzoate on ATPase activity of myosin were also studied. All these three myosin properties are very characteristic when the myosin from a fast muscle is compared with the myosin from a slow muscle of the same animal, but no relationship between these three myosin properties and ATPase activity of myosin was found, when homologous muscles of various mammals were compared.
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PMID:Myosin from fast and slow skeletal and cardiac muscles of mammals of different size. 12 84

The intracellular location of the binding site of antibody against purified myosin prepared from equine leucocytes was investigated in neutrophils and lymphocytes by electron microscopy using peroxidase-labelled antibody method. The myosin extracted from equine leucocytes could bind skeletal muscle F-actin and the formed complex showed the biophysical and biochemical properties and electron microscopic appearance of actomyosin. On immunodiffusion, the leucocyte myosin formed a single precipitin line with its antibody prepared in rabbits. The antibody also formed single precipitin lines with myosins from lymphocytes and thrombocytes, fusing with each other. The antibody against the leucocyte myosin did not react with myosins from skeletal or arterial smooth muscle. The specificity of the antibody was further established by determination of K+-EDTA-activated ATPase activity remained in the supernate of antigen-antibody mixture. Under electron microscope, the intracellular immunoreactive products of peroxidase labelled antibody were found in cytoplasm of neutrophils and lymphocytes incubated with antibody against leucocyte myosin, but not in neutrophils or lymphocytes treated with IgG from normal rabbits.
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PMID:Leucocyte myosin and its location in the cell. 12 83

A new technique for obtaining a myofibril-like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate-gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca2+ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin-like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca2+-sensitivity in tropomyosin-free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.
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PMID:Preparation and properties of vertebrate smooth-muscle myofibrils and actomyosin. 12 55

The effect of F-actin upon the binding of ADP to rabbit skeletal muscle myosin, heavy meromyosin, and subfragment 1 was studied by equilibrium dialysis, ultracentrifuge transport, and light scattering techniques. Both myosin and H-meromyosin (HMM) bind a maximum of approximately 1.6 mol of ADP/mol of protein, while S-1 binds approximately 0.9 mol of ADP/mol of protein. The affinity for ADP of all three preparations was similar at a given ionic strength (approximately 10(6) M-1 at 0.05 M KCl) and decreased with increasing ionic strength. Under conditions similar to those used for the measurement of ADP binding, the binding sites of myosin, HMM, and subfragment 1 (S-1) are saturated with actin at molar ratios of 2, 2, and 1 mol of actin monomer/mol of protein, respectively, as determined by light scattering, ultracentrifuge transport, and in the case of myosin by ATPase measurements. F-actin was found to inhibit ADP binding, but even at an actin concentration at least twice that required for saturation of myosin, HMM, or S-1, significant ADP binding remained. This ADP binding was inhibited by 10(-4) M pyrophosphate. The observations are consistent with the formation of an actomyosin-ADP complex in which actin and ADP are bound to myosin at distinct but interacting sites.
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PMID:Effect of F-actin upon the binding of ADP to myosin and its fragments. 12 42

When ATP binds to myosin in the presence of Mg2+ there follows a rapid cleavage reaction to yield a myosin-product complex whose breakdown is rate-limiting in the overall adenosine triphosphatase reaction at 21 degrees and pH 8.0. Recent kinetic studies on this system have led to the proposal that the cleavage of ATP bound to myosin is reversible. This conclusion is based in part on the observation that when ATP is mixed with an excess of myosin active sites a small amount of tightly bound ATP exists whose life-time coincides with that of the myosin-product complex and implies these two species are in equilibrium during their decay. Previous oxygen exchange studies have shown that phosphate released as free product contains more than one oxygen atom from water. A rapid equilibration between myosin-bound ATP and a myosin-products complex can account for the extra water oxygen incorporation of the product phosphate. Such a model requires that the gamma-phosphoryl group of the bound ATP also exchanges its oxygen atoms with water. Results presented in this paper show that protein-bound ATP labeled in the three terminal oxygen atoms of the gamma-phosphoryl group with 18O exchanges about 75% of its label within 2 s of binding to the active site of myosin. This result provides chemical evidence for a model in which bound ATP undergoes a reversible reaction with water. Incomplete exchange may arise from kinetic and/or structural restraints on the mechanism and plausible models are discussed.
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PMID:Oxygen exchange in the gamma-phosphoryl group of protein-bound ATP during Mg2+-dependent adenosine triphosphatase activity of myosin. 12 49

1. The dependence on ATP concentration of ATPase activity and light scattering decrease of acto-HMM could be described at very low ionic strength by one hyperbolic adsorption isotherm with a dissociation constant of 3 X 10(-6)M. Hence the increase of ATP ase activity was paralleled by a decrease in light scattering. At higher values of ionic strength ATPase activity stopped rising before HMM was completely saturated with ATP. Higher ionic strength prevented ATPase activity from further increasing when the rigor links (links between actin and nucleotide-free myosin), which have formerly protected the ATPase against the suppressing action of higher ionic strength have fallen below a certain amount. This protecting influence of rigor links did not require tropomyosin-troponin. 2. For complete activation of ATPase activity by actin less actin was needed when HMM was incompletely saturated with ATP than when it was completely saturated with ATP. 3. The apparent affinity of ATP to regulated acto-HMM (which contained tropomyosin-troponin) was lower than to unregulated acto-HMM (which was devoid of tropomyosin-troponin). In the presence of rigor complexes (indicated by an incomplete decrease of light scattering) the ATPase activity of regulated acto-HMM was higher than that of unregulated acto-HMM. At increasing ATP concentrations the ATPase activity of regulated acto-HMM stopped rising at a similar degree of saturation with ATP as the ATPase activity of unregulated acto-HMM at the same ionic strength.
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PMID:ATPase activity and light scattering of acto-heavy meromyosin: dependence on ATP concentration and on ionic strength. 12 82

Changes in the mono- and divalentcation-stimulated ATPase activities of myosin progressively labeled with N-ethyl-[2,3-14C2]-maleimide were used to classify the readily reacting thiol groups into 3 types. The results show that one thiol-1 and one thiol-2 group are associated with each of the 2 active sites of myosin. Concentrations of KCl higher than 0.4M and/or temperatures above 10 degrees C lead to exposure of a variable number of thiol groups of a third class not affecting the enzymic properties. Although modification of thiol groups itself results in changes in structure and function of the protein, the patterns of incorporation of N-ethyl-[14C2]-malemide under various conditions of temperature, ionic strength and ligands bound to the protein revealed 9 different conformations of intact myosin. These were distinguished on the basis of the relative reactivity of the 3 different classes of thiol groups. The sequence of blockage of thiol groups reveals that cooperativity between the 2 active sites is induced by binding of a magnesium nucleotide complex to the protein. In the conformation of the long-lived myosin-product intermediate occuring during hydrolysis of Mg-ATP at 25 degrees C, 4 thiol groups of the third class react as well as or even more readily than those of the first and second classes.
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PMID:Conformational differences in myosin, IV.[1-3] Radioactive labeling of specific thiol groups as influenced by ligand binding. 12 41


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