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)

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

Calcium-dependent distance changes have been determined by resonance energy transfer in binary and ternary troponin complexes in order to collect evidence for the structural rearrangements which are part of the hypothetical trigger mechanism of skeletal muscle contraction. Donor and acceptor fluorophores were either intrinsic tryptophans in subunits with a favourable sequence from different species, quasi-intrinsic Tb3+ ions bound to troponin C or extrinsic labels attached to specific cysteine or methionine residues. All chemically modified subunits proved fully active in conferring calcium sensitivity onto myosin ATPase. Nine distances were determined between five sites which allowed construction of a three-dimensional lattice representing the spatial distribution of four sites in the ternary complex of troponin C, I and T. Distances in binary complexes were nearly unaltered upon addition of the third subunit. Regulatory calcium binding caused distance changes of the order of 0.7-1.1 nm. In view of the large displacements of the hypothetical mechanism, they turned out to be smaller than anticipated. The fluorophoric sites selected may be localized in a zone of the troponin complex which happens to be relatively little affected by the mechanism. Alternatively, amplification of the moderate changes seen here would require the complete set of thin filament proteins.
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PMID:Calcium-dependent distance changes in binary and ternary complexes of troponin. 235 Nov 40

A substance which inhibits the myosin ATPase has been detected from an Okinawan marine sponge. The inhibitor has been isolated from the methanol-soluble extract of the sponge by gel filtration and hydrophobic chromatography. The isolated inhibitor is an amphipathic peptide, which is rich in Asp, Glu, Ser, and Gly, and devoid of Met and Trp. The molecular weight of the peptide is about 6300 as determined by gel filtration, amino acid analysis, and sodium dodecyl sulfate-gel electrophoresis. The peptide is a potent inhibitor not only for the K+-, Ca2+-, and Mg2+-ATPases of myosin, its subfragment-1, and actomyosin, but also for superprecipitation of actomyosin, inhibiting them completely in the range of 10-400 ng/ml. The peptide inhibitor may provide a useful tool to elucidate the structure-function relationship of the myosin ATPase.
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PMID:A novel peptide inhibitor of the myosin ATPase from an Okinawan marine sponge. 253 Feb 20

One of the leading causes of mortality in diabetics is myocardial disease. In the past few years this subject has generated a significant amount of interest with the result that myocardial problems associated with diabetes are far better understood. Though originally thought to occur as a result of atherosclerosis, various studies have shown that heart disease can occur in the absence of atherosclerosis, suggesting a diabetic cardiomyopathy. Using diabetic animals, it has been possible to characterize diabetes-induced myocardial abnormalities. Diabetic rat hearts do not respond to conditions of high stress as well as controls. The functional depression is accompanied by altered cardiac enzyme systems. A decrease in myosin ATPase activity which appears to be a result of diabetes-induced hypothyroidism is seen. Also, a depression of sarcoplasmic reticular calcium ATPase, along with a depression of calcium uptake by the SR, is seen in diabetic rat hearts. Na+, K+ ATPase activity has also been shown to be depressed and the depression appears to correlate with depressed atrial contractility. High levels of circulating fats in diabetics may alter the integrity of membranes leading to altered enzyme activities. Insulin treatment has been relatively successful at reversing or preventing myocardial changes in the diabetic rat. Other treatments that have been studied include thyroid hormone treatment, since the depression of myosin ATPase can be corrected by such treatment; and carnitine treatment, as the elevation of long chain acyl carnitines (LCAC) and the resulting depression of calcium uptake in the SR can be so normalized. These treatments have not been successful at normalizing cardiac function. A combination of the two treatments normalized function only partially, suggesting that factors besides myosin ATPase and SR calcium uptake are involved. Other treatments that have been tried include vanadate, methyl palmoxirate, and choline and methionine. Vanadate treatment has proved to be encouraging in that it normalizes both function and hyperglycemia. Methyl palmoxirate, a fatty acid analog, normalized only the elevation of LCAC but did not affect function. Methionine and choline were only partially successful in preventing the functional alterations of diabetic rat hearts. The purpose of the present article is to review our understanding of diabetes-induced myocardial problems and their possible causes. Findings from our laboratory and others are described in which attempts have been made to normalize cardiac function.
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PMID:Diabetes-induced abnormalities in the myocardium. 293 41

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

Although the alkali or essential light chains of skeletal muscle myosin are not required for actin-activated myosin ATPase activity, these myosin subunits are necessary for force transmission with in vitro actin motility assays and are believed to stabilize the alpha-helical neck region of myosin subfragment-1. To probe the functions of the essential light chains during myofibril assembly, we used recombinant DNA procedures to deplete this light chain in cultured muscle. Retroviral expression vectors were constructed which encoded the exon-1 sequence of the myosin light chain-1 gene in antisense orientation. These vectors were applied to myogenic cells from embryonic chick and quail pectoralis muscle. Colonies expressing antisense RNA were selected in growth medium containing the neomycin analogue G-418, plus 5-bromo-2'-deoxyuridine (BrdU) and triggered to differentiate by removal of the latter. Expression of antisense myosin light chain-1 mRNA impaired muscle development. In the antisense cultures there were more mononucleated cells, fewer and smaller myotubes which had poorly developed myofibrils and high levels of diffusely staining myosin heavy chain, not apparent in control myotubes. Protein synthesis in the myotube cultures was analyzed by 35S-methionine labelling and two-dimensional gel electrophoresis. Except for a suppression of approximately 80% of myosin light chain-1f synthesis, the overall pattern of protein synthesis was not altered significantly. These studies suggest that retardation of myosin light chain-1f accumulation inhibits or delays myofibrillogenesis.
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PMID:Antisense suppression of skeletal muscle myosin light chain-1 biosynthesis impairs myofibrillogenesis in cultured myotubes. 775 4

We have examined the structural and functional effects of site-directed methionine oxidation in Dictyostelium (Dicty) myosin II using mutagenesis, in vitro oxidation, and site-directed spin-labeling for electron paramagnetic resonance (EPR). Protein oxidation by reactive oxygen and nitrogen species is critical for normal cellular function, but oxidative stress has been implicated in disease progression and biological aging. Our goal is to bridge understanding of protein oxidation and muscle dysfunction with molecular-level insights into actomyosin interaction. In order to focus on methionine oxidation and to facilitate site-directed spectroscopy, we started with a Cys-lite version of Dicty myosin II. For Dicty myosin containing native methionines, peroxide treatment decreased actin-activated myosin ATPase activity, consistent with the decline in actomyosin function previously observed in biologically aged or peroxide-treated muscle. Methionine-to-leucine mutations, used to protect specific sites from oxidation, identified a single methionine that is functionally sensitive to oxidation: M394, near the myosin cardiomyopathy loop in the actin-binding interface. Previously characterized myosin labeling sites for spectroscopy in the force-producing region and actin-binding cleft were examined; spin-label mobility and distance measurements in the actin-binding cleft were sensitive to oxidation, but particularly in the presence of actin. Overall secondary structure and thermal stability were unaffected by oxidation. We conclude that the oxidation-induced structural change in myosin includes a redistribution of existing structural states of the actin-binding cleft. These results will be applicable to the many biological and therapeutic contexts in which a detailed understanding of protein oxidation as well as function and structure relationships is sought.
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PMID:Structural and functional impact of site-directed methionine oxidation in myosin. 2198 99