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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 negatively charged residues in the N-terminus of actin and the 697-707 region on myosin subfragment 1 (S-1), containing the reactive cysteines SH1 and SH2, are known to be important for actin-activated myosin ATPase activity. The relationship between these two sites was first examined by monitoring the rates of SH1 and SH2 modification with N-ethylmaleimide in the presence of actin and, secondly, by testing for direct binding of SH1 peptides to the N-terminal segment on actin. While actin alone protected SH1 from N-ethylmaleimide modification, this effect was abolished by an antibody against the seven N-terminal amino acids on actin, F(ab)(1-7), and was greatly reduced when the charge of acidic residues at actin's N-terminus was altered by carbodiimide coupling of ethylenediamine. Neither F(ab)(1-7) nor ethylenediamine treatment reversed the effect of F-actin on SH2 reactivity in SH1-modified S-1. These results show a communication between the SH1 region on S-1 and actin's N-terminus in the acto-S-1 complex. To test whether such a communication involves the binding of the SH1 site on S-1 to the N-terminal segment of actin, the SH1 peptide IRICRKG-NH2(4+) was used. Cosedimentation experiments revealed the binding of three to six peptides per actin monomer. Peptide binding to actin was affected slightly, if at all, by F(ab)(1-7). The antibody also did not change the polymerization of G-actin by the peptides. The peptides caused a small reduction in the binding of S-1 to actin and did not change the binding of F(ab)(1-7).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Antibody and peptide probes of interactions between the SH1-SH2 region of myosin subfragment 1 and actin's N-terminus. 142 Feb 4

Scallop adductor myosin is regulated by its subunits; the regulatory light chain (R-LC) and essential light chain (E-LC). Myosin light chains suppress muscle activity in the absence of calcium and are responsible for relaxation. The binding of Ca2+ to the myosin triggers contraction by releasing the inhibition imposed on myosin by the light chains. To map the functional domains of the R-LC, we have carried out mutagenesis followed by bacterial expression. Both wild-type and mutant proteins were hybridized to scallop myosin heavy chain/E-LC to map the regions of the light chain that are responsible for the binding to the myosin heavy chain/E-LC, for restoring the specific calcium-binding site, and controlling the myosin ATPase activity. The R-LC is expressed in Escherichia coli using the pKK223-3 (Pharmacia) expression vector and has been purified to greater than 90% purity. E. coli-expressed wild-type R-LC differs from the native R-LC by having the initiating methionine residue and an unblocked NH2 terminus. The wild-type R-LC restores Ca2+ binding and Ca2+ sensitivity when hybridized to scallop myosin. A point mutation of the sixth Ca2(+)-liganding position of domain I (Asp39----Ala39) results in a R-LC that binds more weakly to the heavy chain/E-LC and restores the specific Ca2(+)-binding site but not regulation of the actin-activated Mg2+ ATPase. A second mutation was produced by substituting the last 11 residues of the COOH terminus with 15 different residues. This mutant restores the specific Ca2(+)-binding site, but does not restore Ca2+ regulation to the actin-activated ATPase activity. Several other point mutations do not alter light chain function. The experiments directly establish that the divalent cation-binding site of domain I is functionally distinct from the specific Ca2(+)-binding site. The results indicate that an intact domain I and the COOH terminus are required to suppress the myosin ATPase activity. The fact that the domain I mutation and the COOH-terminal mutation disrupt regulation but do not affect Ca2(+)-binding indicates that these two aspects of regulation are separable and, therefore, the R-LC has distinct functional regions.
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PMID:Regulation of scallop myosin by mutant regulatory light chains. 214 99

Modification of histidine residues, SH- and epsilon-NH2-groups of myosin from rat sarcoma-45 by specific reagents was studied. It was shown that diethylpyrocarbonate modifies histidine residues essential for the ATPase activity. A kinetic analysis of myosin epsilon-NH2-groups modification by 2,4,6-trinitrobenzene sulfonate revealed that myosin trinitrophenylation and its inactivation by Ca2(+)-ATPase occurs in two steps: a fast and a slow (Km = 2400 and 1.7 s-1 M-1, respectively). Two essential epsilon-NH2-groups of tumour myosin active sites react in the fast reaction. The relatively low concentrations of p-chloromercuribenzoic acid activate rat sarcoma-45 myosin Ca2(+)-ATPase and Mg2(+)-ATPase, whereas higher ones inhibit the enzyme. The data obtained suggest that two SH-groups, SH1 and SH2 are essential for the tumour myosin ATPase function.
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PMID:[Study of the modification of histidine residues, SH- and epsilon-NH2-groups of rat sarcoma-45 myosin by specific reagents]. 215 Mar 35

The heavy chain of myosin from rabbit skeletal muscle can be cleaved at three sites by irradiation with near-ultraviolet light in the presence of 0.1-1.0 mM vanadate. The sigmoidal dependence upon vanadate concentration, with half-maximal rate occurring at about 0.5 mM vanadate and a sigmoidicity of 2.7, is consistent with the chromophore responsible for cleavage being oligomeric vanadate. Cleavage occurs at two sites located within the head region of the molecule, 23 kDa and 75 kDa from the NH2-terminus; these sites are cleaved equally well in heavy meromyosin and subfragment 1. In the presence of 1 mM vanadate, the half-times for cleavage of the 23-kDa and 75-kDa sites are about 15 and 10 min, respectively. The rate of cleavage at both these sites is retarded 2-3-fold by the presence of greater than 10 microM MgATP. The third photocleavage site is located about 5-10 kDa from the COOH terminus of the intact heavy chain, and cleaves equally well in the isolated rod and in light meromyosin. Cleavage at this site occurs with a half-time of 138 min, and its rate is unaffected by the presence of MgATP. The vanadate-mediated cleavage of the heavy chains is accompanied by characteristic changes in the myosin ATPase properties, with the Ca2+, Mg2+ and actin-activated Mg2+ ATPases becoming elevated, whereas the K+/EDTA ATPase becomes inactivated. The sites of photocleavage in the myosin heavy chain might be associated with sites of phosphate binding.
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PMID:Vanadate-mediated photocleavage of rabbit skeletal myosin. 253 8

Various aspects of actin--myosin interaction were studied with actin preparations from two types of smooth muscle: bovine aorta and chicken gizzard, and from two types of sarcomeric muscle: bovine cardiac and rabbit skeletal. All four preparations activated the Mg2+-ATPase activity of skeletal muscle myosin to the same Vmax, but the Kapp for the smooth muscle preparations was higher. At low KCl, pH 8.0 and millimolar substrate concentrations the Kapp values differed by a factor of 2.5. This differential behaviour of the four actin preparations correlates with amino acid substitutions at positions 17 and 89 of actin polypeptide chain, differentiating the smooth-muscle-specific gamma and alpha isomers from cardiac and skeletal-muscle-specific alpha isomers. This correlation provides evidence for involvement of the NH2-terminal portion of the actin polypeptide chain in the interaction with myosin. The differences in the activation of myosin ATPase by various actins were sensitive to changes in the substrate and KCl concentration and pH of the assay medium. Addition of myosin subfragment-1 or heavy meromyosin in the absence of nucleotide produced similar changes in the fluorescence of a fluorescent reagent N-(1-pyrenyl)-iodoacetamide, attached at Cys-374, or 1,N6-ethenoadenosine 5'-diphosphate substituted for the bound ADP in actin protomers in gizzard and skeletal muscle F-actin. The results are consistent with an influence of the amino acid substitutions on ionic interactions leading to complex formation between actin and myosin intermediates in the ATPase cycle but not on the associated states.
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PMID:Identification of amino acid substitutions differentiating actin isoforms in their interaction with myosin. 293 50

Several regions within the 35-kDa COOH-terminal portion of caldesmon have been implicated in the ability of caldesmon to inhibit actin-activated myosin ATPase activity. To further define the functional regions of caldesmon, we have studied the effects of three chymotryptic fragments, one fragment produced by CNBr digestion and two fragments produced by digestion with submaxillaris arginase C protease, on the relaxed stiffness and active force of rabbit psoas fibers. Each of the regions of caldesmon studied had either direct or indirect effects on single-fiber mechanics. The 35-kDa and 20-kDa fragments of caldesmon, like intact caldesmon, were effective inhibitors of fiber stiffness, a measure of cross-bridge attachment. The 7.3-kDa and 10-kDa fragments, which constitute the NH2 and COOH halves of the 20-kDa fragment, inhibited both relaxed fiber stiffness and active force production, but with a reduced efficacy compared to the 20-kDa fragment. These results suggest that several regions within the 35-kDa COOH-terminal region of caldesmon are required for optimum function of caldesmon and that function includes inhibition of weak cross-bridge attachment and force production.
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PMID:Inhibition of cross-bridge binding to actin by caldesmon fragments in skinned skeletal muscle fibers. 913 74

Caldesmon inhibits the activation of myosin ATPase activity by actin-tropomyosin. Caldesmon also inhibits the binding of myosin to actin. There is disagreement as to the degree to which competitive displacement of myosin subfragment binding to actin is responsible for the inhibition of ATPase activity. We have examined the possibility that one or more molecules of S1 may bind to actin-tropomyosin-caldesmon without having the normal actin activation of ATPase activity. The effect of caldesmon on the binding and ATPase activity of S1 was measured at several initial levels of saturation of S1 to determine if a fraction of the bound S1 was resistant to displacement by caldesmon. In the case of both unmodified S1 and rhoPDM-modified S1, most, but not all, of the S1 was displaced by caldesmon. The results are consistent with a single molecule of S1 binding with low affinity for each seven actin monomers that are fully saturated with caldesmon and tropomyosin. This single S1 is not necessarily bound directly to actin but may be attached to the NH2-terminal region of caldesmon.
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PMID:Caldesmon-actin-tropomyosin contains two types of binding sites for myosin S1. 958 67