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)

Myosin was extracted from normal human hearts (autopsy material) and compared to that of pig heart and rabbit white skeletal muscle. Myosin light subunits were isolated by a preparative urea gel electrophoresis. These subunits were shown by urea and sodium dodecylsulfate gel electrophoresis to be only slightly affected by the time lapse between death and the beginning of myosin extraction. This was also true for myosin ATPases. The Ca-2+-activated ATPases of pig and human heart myosins have the same apparent Km and V, whereas white skeletal muscle myosin ATPase has the same Km with a higher V. Human myosin light subunits, when compared to those of pig heart possess: (i) different molecular weights: 27 999 and 18 000 datlons for pig heart, and 25 000 and 19 000 daltons for human heart. (ii) for both the light chains, different ultraviolet spectra and a higher helical content for the subunit molecular weight 25 000. (iii) a different composition for several amino acids (Tyr, Pro, Lys). A third light subunit (molecular weight 15 000) was occasionally seen in human as well as pig heart myosin. It concentration varied inversely with that of the subunit molecular weight 27 000-25 000, and so was probably a degradation product of the heaviest subunit.
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PMID:Human cardiac myosin ATPase and light subunits. A comparative study. 12 84

Increased afterload causes increased cardiac myosin synthesis and ultimately leads to hypertrophy. Since the latter is associated with altered myosin ATPase activity, it was of interest to study the synthesis of myosin subunits in the acute response to this stress. An in vitro guinea pig heart preparation was used which allowed application of afterload to the right ventricle with unaltered coronary flow, and also permitted measurement of synthesis of myosin heavy chains (HC) and combined light chains (LC) by continuous perfusion with labelled amino acids (3H-lysine and/or 3H-phenylalanine) of constant specific activity. Isolation of 3H-labelled HC and LC with heterologous unlabelled carrier was possible because of identical mobilities of HC's and LC's from unlabelled lamb carrier myosin and 3H-labelled guinea pig myosin. This permitted study of comparative synthesis of the HC and LC in small samples as the single guinea pig right ventricle (100--150 mg) and avoided errors inherent in pooling hearts or in measurement of turnover in the nonsteady state. After 3 h or perfusion, the ratio of synthesis of HC/LC was 2 : 1 in controls. This ratio increased significantly to 3 : 1 in after load. It is possible that the disproportionate increase in HC synthesis may lead to stoichiometric problems in myosin assembly which ultimately effect altered myosin ATPase activity.
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PMID:Synthesis of myosin heavy and light chains in the afterloaded guinea pig right ventricle. 75 24

The heptapeptide Ile-Arg-Ile-Cys-Arg-Lys-Gly-OEt is the analog of the S-site, one of the actin-binding sites in myosin [Suzuki et al. (1987) J. Biol. Chem. 262, 11410-11412]. Various substituted heptapeptides were synthesized, and the dissociation constants of each acto-heptapeptide complex was measured. Comparison of the dissociation constants indicated that the hydrophobic side chain of Ile-1 was critical for the binding with F-actin, but not that of Ile-3. The positive charge and the side chain length of Arg-2 were also important. The presence of a sulfur atom in the Cys-4 was also necessary. The affinity of the N-terminal Ile-Arg-Ile part for F-actin was influenced by the kind of residues in the C-terminal tetrapeptide part. Based on these results, the side chains of Ile(702), Arg(703), and Cys(SH1)(705) in myosin subfragment-1 heavy chain were assigned to be critical for the binding with F-actin. The amino acid sequence of S-1 heavy chain containing these critical residues for the S-site from residue number 700 to 717 can be predicted as an analogue of the segment B of the ATP-binding site [Walker et al. (1982) EMBO J. 1, 945-951]. The actin-binding S-site possibly shares a part of the ATP-binding site in myosin. We discuss the possibility that the S-site is an inhibitory site of myosin ATPase and the so-called actin-activation of myosin ATPase is a deinhibition induced by transient binding of F-actin to the S-site.
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PMID:Roles of the amino acid side chains in the actin-binding S-site of myosin heavy chain. 214 38

Three kinds of ATP analogues were synthesized. These ATP analogues can be classified into two conformations, i.e. syn and anti forms with respect to the N-glycosidic bond between adenine and ribose groups of ATP. 3'-O-(N-Methylanthraniloyl)-2-azidoadenosine 5'-triphosphate (MantN2(3)ATP) is recognized as the anti form, as ATP, and the other two, 3'-O-(N-methylanthraniloyl)-8-azidoadenosine 5'-triphosphate (MantN8(3)ATP) and 1,N6-etheno-8-azidoadenosine 5'-triphosphate (epsilon N8(3)ATP) are both syn forms. Mant and etheno groups are both fluorescent which allows detection of their binding to proteins. The photochemical binding of azido groups in ATP analogues to the myosin active site, examined in the presence and absence of ATP, showed that all the analogues bound to the site of myosin ATPase. These analogues also acted as substrates of the ATPase and were hydrolyzed in the active site, as judged by competitive inhibition of the ATPase and by their ATPase activities. Of these analogues, MantN2(3)ATP is very similar to ATP in divalent-cation dependence of its hydrolysis rate and in its ability to trap ADP in the active site with vanadate, while the other two are different from ATP in these respects. The photochemical binding sites of ATP analogues were localized by gel electrophoresis of trypsinized myosin ATPase with photocross-linked ATP analogues and/or by isolating the modified peptides. MantN2(3)ATP was found in the 23-kDa fragment which has a structure common to ATP-binding proteins, i.e. Gly-Xaa-Xaa-Gly-Xaa-Gly-Lys-Thr. Mant N8(3)ATP was found in a region of the 20-kDa fragment where actin is reported to attach.
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PMID:Localization of the ATP-binding site in the 23-kDa and 20-kDa regions of the heavy chain of the skeletal muscle myosin head. 252 53

Cultured neonatal rat heart cells are a useful model for studying the regulation of myocyte growth. The myosin content of heart cells increases between days 1 and 4 in culture. To determine if contraction per se can regulate myocyte growth, myosin content and protein synthesis were compared in spontaneously contracting and noncontracting cultured heart cells. Myosin content, assayed as the total myosin ATPase activity per culture dish, was significantly increased in contracting cells after 3, 4, and 5 days in culture. Protein synthesis was measured by incorporation of [14C]lysine into total cell protein and into sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved myosin. Contraction stimulated both total cell protein content and protein synthesis by day 3 in culture. Compared with heart cells arrested with 50 mM KCl, myosin synthesis was significantly increased by 96, 112, and 46% at days 2, 3, and 4, respectively. Similar results were observed when myosin content and protein synthesis in contracting myocytes were compared with cells arrested with either 25 mM KCl or 10(-5) M verapamil. The present studies suggest that contraction increases the myosin content in cultured heart cells and that this increase is mediated via a stimulation of myosin synthesis in association with cell growth.
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PMID:Contraction regulates myosin synthesis and myosin content of cultured heart cells. 293 98

Solution properties of skeletal muscle actin, modified at lysine-61 with fluorescein isothiocyanate (FITC) [Burtnick, L.D. (1984) Biochim. Biophys. Acta 791, 57-62], were re-examined in this work by light scattering, analytical ultracentrifugation, fluorescence, electron microscopy and myosin ATPase activity measurements. Fluorescence measurements using trace amounts of actin labeled with N-(1-pyrenyl)iodoacetamide showed that the FITC modification inhibited but did not block completely the polymerization of actin by KCl and MgCl2. Sedimentation velocity runs of FITC-actin, incubated with 100 mM KCl and 2 mM MgCl2, revealed the presence in these solutions of polymeric, oligomeric and monomeric species. The critical concentration for FITC-actin polymerization under these conditions was 12 microM. As judged by electron microscopy, FITC-actin polymers were similar to but generally shorter than standard F-actin filaments. Light scattering measurements indicated that FITC modification inhibited also the polymerization of actin by myosin subfragment 1 (S1) but the resulting complexes were indistinguishable from standard, decorated actin filaments. MgATPase measurements showed that FITC-actin, polymerized by preincubation with S1, activated the MgATPase activity of S1 while the monomeric labeled protein did not. Thus, in analogy to native actin, the activating function of FITC-actin depended on the formation of actin filaments. Results presented in this study suggest that the region around lysine-61 of actin plays an important role in actin-actin contact and is less crucial to actomyosin interaction.
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PMID:Polymerization of actin modified with fluorescein isothiocyanate. 296 82

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

We have used isotope-edited nuclear magnetic resonance spectroscopy, binding studies, and ATPase activity assays to investigate the interaction with F-actin of the 10 kDa C-terminal 658C fragment of chicken gizzard caldesmon and two site-directed mutants of this fragment. Simultaneous dual-sited contacts with F-actin are observed for the segments of the 658C sequence flanking tryptophan residues 692 and 722. Competition experiments showed that both 658C contacts with actin are displaced by substoichiometric concentrations of the short inhibitory region of troponin-I indicative of different binding sites on actin for these regions of troponin-I and caldesmon. Substitution of caldesmon serine-702 by aspartic acid within the spacer region linking the two actin contacts of 658C led to weaker binding but with retention of equivalent affinity for each interaction site. Differential binding affinity of the two sites was achieved by replacement of the sequence Glu691-Trp-Leu-Thr-Lys-Thr696 by Pro-Gly-His-Tyr-Asn-Asn. Consistent with these data, the concentration of this Cg1 mutant required to achieve 50% inhibition of actin-tropomyosin-activated myosin ATPase was 4-fold greater than found for the 658C fragment. Although calmodulin binding to Cg1 was observed, calmodulin proved ineffective in relieving the inhibition induced by the binding of this mutant to actin. These results are discussed in light of the actin contacts which are involved in the inhibitory activity possessed by different regions of the C-terminus of caldesmon.
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PMID:Structure-activity studies of the regulatory interaction of the 10 kilodalton C-terminal fragment of caldesmon with actin and the effect of mutation of caldesmon residues 691-696. 948 78

Actin binding to skeletal muscle myosin subfragment-1 (S1) increases the dissociation rate of reaction products from the myosin ATPase site; conversely, ATP binding facilitates dissociation of complexed acto-S1. However, details of the molecular mechanism by which the ATP- and actin-binding sites communicate with each other is still obscure. We present evidence that the effect of actin is mediated by a conformational change in the loop containing amino acids from 677 to 689 [loop M (677-689)], a segment of the 20-kDa tryptic fragment that contributes to the structure of the ATP-binding cleft. Initially, a fluorescent ADP analogue, methylanthranyloyl-8-azido-ADP (Mant-8-N3-ADP), was covalently crosslinked to loop M (Mant-S1), perhaps at Lys 681. Actin-activated Mg2+-ATP hydrolysis by Mant-S1 was accelerated approximately 6 times over that by unmodified S1, suggesting that the ATPase site is not blocked by the ADP analogue crosslinked in the loop M (677-689). Nevertheless, analysis of Mant-group fluorescence polarization and acrylamide-induced quenching showed the crosslinked probe to be entrapped within the ATP-binding cleft at a location where Mant-group rotational mobility was hindered, and where it was relatively inaccessible to the solvent. Exposing Mant-S1 to Mg2+-ATP and/or actin elicited similar decreases in fluorescence polarization, indicating increased rotational mobility of the Mant-group and movement of crosslinked Mant-8-N3-ADP to a less hindered position. Stern-Volmer quench curves showed that Mant-8-N3-ADP was translocated to a site where it was more accessible to dissolved quencher, perhaps outside the ATP-binding cleft. Since actin does not bind to the ATPase site, actin-induced translocation of Mant-8-N3-ADP crosslinked to loop M (677-689) probably results from a conformational change in loop M (677-689). These results suggest that loop M acts as a signal transducer mediating communication between the ATP- and actin-binding sites.
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PMID:A unique loop contributing to the structure of the ATP-binding cleft of skeletal muscle myosin communicates with the actin-binding site. 972 61

The present study investigated the role of actin polymerization and myosin motor protein activity in the gliding motility of Cryptosporidium parvum sporozoites. Short motility trails were detected using an indirect immunofluorescent assay (IFA) with a polyclonal antisporozoite antibody following incubation of sporozoites on poly-L-lysine-coated glass slides. Sporozoite motility was blocked following exposure to cytochalasin D, a myosin light-chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexhydro-1,4-diazapin e, and the myosin ATPase inhibitor 2,3-butanedione monoxime. Sporozoites were observed to form rounded, blunt-ended shapes when exposed to these same inhibitors. Incubation of purified oocysts with these compounds did not significantly inhibit in vitro excystation or subsequent infectivity in cultured epithelial cells. Indirect IFA revealed a uniform distribution of actin protein throughout the body of the sporozoite; immunoelectron microscopy confirmed a diffuse intracellular pattern of gold particles in excysted sporozoites. Collectively, these findings show that sporozoite motility is dependent upon an intact actin-myosin motor system, and the dynamic interaction of F-actin and myosin motor proteins has a further role in maintaining the structural integrity of excysted sporozoites. Further, in vitro excystation and infectivity of C. parvum occurs in the absence of dynamic sporozoite locomotion.
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PMID:Actin-dependent motility in Cryptosporidium parvum sporozoites. 979 29


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