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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The two cardiac myosin heavy chain isoforms, alpha and beta, differ functionally, alpha Myosin exhibits higher actin-activated ATPase than does beta myosin, and hearts expressing alpha myosin exhibit increased contractility relative to hearts expressing beta myosin. To understand the molecular basis for this functional difference, we determined the complete nucleotide sequence of full-length rat alpha and beta myosin heavy chain cDNAs. This study represents the first opportunity to compare full-length fast ATPase and slow ATPase muscle myosin sequences. The alpha and beta myosin heavy chain amino acid sequences are more related to each other than to other sarcomeric myosin heavy chain sequences. Of the 1938 amino acid residues in alpha and beta myosin heavy chain, 131 are non-identical with 37 non-conservative changes. Two-thirds of these non-identical residues are clustered, and several of these clusters map to regions that have been implicated as functionally important. Some of the regions identified by the clusters of non-identical amino acid residues may affect actin binding, ATP hydrolysis and force production.
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PMID:Full-length rat alpha and beta cardiac myosin heavy chain sequences. Comparisons suggest a molecular basis for functional differences. 261 40

High-performance ion-exchange chromatography of myosin using a DEAE-5PW packing was used to purify myosin from skeletal, cardiac and smooth muscle. This method produces high-speed resolution (30-min analysis) of myosin from contaminating myofibrillar proteins. The column has a high capacity for binding myosin (up to 1 g) and can be used for small-scale preparation of highly purified myosin. Gel analysis in the presence of sodium dodecyl sulfate showed recovery of myosin with very little contamination of other myofibrillar proteins. Myosin was also recovered from small biopsy samples (0.1 g) by a direct extraction technique with recovery of biological ATPase activity.
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PMID:High-performance ion-exchange chromatography of myosin using a DEAE-5PW column. 279 83

Myosin was reacted with 2,4,6-trinitrobenzene sulphonate (TNBS) in the presence or absence of Mg-pyrophosphate. The reaction led to trinitrophenylation of lysyl residues which could be divided on the basis of the reaction into three classes: (i) two rapidly reacting lysyl residues (RLR), one residing on each head of myosin, whose rate of reaction depends on the presence of Mg-pyrophosphate; (ii) two lysyl residues which react with intermediate rate (ILR) and reside on the rod segment of myosin; and (iii) the remaining lysyl residues of myosin which react slowly with TNBS. The rate of the trinitrophenylation of RLR was followed spectrophotometrically and enzymatically, measuring an absorbance change at 345 nm, and also changes in K+ (EDTA)-, Mg2+- and Ca2+-activated ATPase activities, respectively. According to analysis of the kinetics of the reaction, Mg-pyrophosphate inhibited the rate of trinitrophenylation in both heads of myosin, not in one head only as was suggested by Miyanishi et al. (J. Biochem Tokyo 85; 1979). Myosin heads (myosin subfragment-1, S-1) were prepared by digesting myosin trinitrophenylated in the absence and presence of Mg-pyrophosphate with chymotrypsin. S-1, with trinitrophenylated RLR, was separated from non-trinitrophenylated S-1 by DEAE cellulose column chromatography. The trinitrophenylated S-1 had a high Mg2+- and a low K+(EDTA)-activated ATPase while the non-trinitrophenylated species had the usual high K+(EDTA)- and low Mg2+-ATPase activity. This results excluded the possibility suggested by Miyanishi et al., that the myosin head, which is resistant to trinitrophenylation in the presence of Mg-pyrophosphate, did not possess K+(EDTA)-activated ATPase activity. The presence of Mg-pyrophosphate during trinitrophenylation substantially affected the enzymic characteristics of the modified myosin. The myosin trinitrophenylated in the presence of Mg-pyrophosphate had a higher K+(EDTA)- and a lower Mg2+-ATPase activity. SH1 (Cys-707) also probably becomes a target of the reaction if myosin is trinitrophenylated in the presence of Mg-pyrophosphate. This is deduced from the following findings: (i) the addition of dithiothreitol after trinitrophenylation partially reversed the loss in the K+(EDTA)-ATPase activity; and (ii) the specific alkylation of the SH1 thiol by 1,5-IAEDANS prior to trinitrophenylation prevented the effect of dithiothreitol on the ATPase activity of myosin. The results indicated that Mg-pyrophosphate induced structural changes in the myosin molecule which influenced the course and possibly the target(s) of trinitrophenylation.
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PMID:The effect of pyrophosphate on the reaction of myosin with 2,4,6-trinitrobenzene sulphonate. 284 63

Age related change of the human minor pectoral muscles was biochemically demonstrated. Myosin-ATPase activity was significantly decreased with age, and was activated with Tris. The degree of the activation by Tris was observed to be lower in old aged patients than in the young. Furthermore, at low concentration of CaCl2 (less than 100 microM), myosin-ATPase activity was higher in the young age than in the old, while at high concentration of CaCl2 (more than 1 mM) no significant difference was observed between young and old age. Decrease with age in activation by primary amine such as Tris would play an important role in the muscle working capacity in old age.
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PMID:Age-related change in activation by Tris (hydroxymethyl) aminomethane on myosin-ATPase activity of human minor pectoral muscles. 293 10

In vertebrate smooth muscles, phosphorylation of the regulatory light chain appears to be necessary for actin activation of the Mg-ATPase activity and for the in vitro assembly of myosin into filaments. From a correlation between the degree of phosphorylation and enzymatic activity, it was suggested that both myosin heads must be phosphorylated before either head could be activated by actin, and that phosphorylation of filamentous myosin occurred in a negatively cooperative manner (Persechini, A., and Hartshorne, D. J. (1981) Science 213, 1383-1385; Ikebe, M., Ogihara, S., and Tonomura, Y. (1982) J. Biochem. (Tokyo) 91, 1809-1812; Sellers, J. R., Chock, P. B., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14181-14188). Here we have determined the mechanism of phosphorylation by separating dephosphorylated and phosphorylated myosin species based on their different structural properties in the minifilament buffer system (5 mM citrate, 22 mM Tris). Fully phosphorylated myosin remained assembled as minifilaments in 1 mM Mg-ATP, but dephosphorylated myosin dissociated to a mixture of folded monomers and dimers. Gel filtration was used to separate these two structures. At intermediate levels of phosphorylation, the relative amount of myosin that formed minifilament and dimer and the degree of phosphorylation of the separated species relative to the initial level of phosphorylation was measured. From these data, it was possible to deduce that singly and doubly phosphorylated myosin remained assembled in the presence of nucleotide. Myosin molecules with 0, 1, or 2 heads phosphorylated could also be separated by nondenaturing gel electrophoresis. The amount of myosin which formed each species was quantitated as a function of phosphorylation. Results from the combined approaches are consistent with a model in which light chain kinase randomly phosphorylates myosin, independent of the state of aggregation of the myosin.
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PMID:Mechanism of smooth muscle myosin phosphorylation. 293 4

To understand the molecular basis of microtubule-associated motility during mitosis, the mechanochemical factors that generate the relevant motile force must be identified. Myosin, the ATPase that interacts with actin to produce the force for muscle contraction and other forms of cell motility, is believed to be involved in cytokinesis but not in mitosis. Dynein, the mechanochemical enzyme that drives microtubule sliding in eukaryotic cilia and flagella, has been identified in the cytoplasm of sea urchin eggs, but the evidence that it is involved in cytoplasmic microtubule-based motility (rather than serving as a precursor for embryonic cilia) is equivocal. Microtubule-associated ATPases have been prepared from other tissues, but their role in cytoplasmic motility is also unknown. Recent work on axoplasmic transport, however, has led to the identification of a novel mechanochemical protein called kinesin, which is thought to generate the force for moving vesicles along axonal microtubules. These results suggest that kinesin may also be a mechanochemical factor for non-axoplasmic forms of microtubule-based motility, such as mitosis. We describe here the identification and isolation of a kinesin-like protein from the cytoplasm of sea urchin eggs. We present evidence that this protein is localized in the mitotic spindle, and propose that it may be a mechanochemical factor for some form of motility associated with the mitotic spindle.
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PMID:Identification of kinesin in sea urchin eggs, and evidence for its localization in the mitotic spindle. 293 90

Myosin was purified rapidly from the nematode Caenorhabditis elegans by an improved method. Crude actomyosin was extracted from the worms at low ionic strength. Paramyosin was removed by repeating the precipitation of myosin filaments in the presence of Mg2+ and the dissolution of them in 0.6 M NaCl. Actin was removed by ultracentrifugation in the presence of Mg-ATP and finally by column chromatography on DEAE-cellulose. This method gave a good yield of myosin (20-30 mg from 50 g wet weight of worms), and its EDTA(K+)-ATPase activity was about 3-fold higher than that of myosin prepared by the method of Harris and Epstein (1979). ATP hydrolysis by nematode myosin showed an initial Pi-burst due to formation of the myosin-phosphate-ADP complex. Tryptophan fluorescence of myosin was enhanced about 8% by ATP. The relationship between the structure and function of myosin is discussed based on the above results and the amino acid sequences of myosins from rabbit skeletal muscle and Caenorhabditis elegans.
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PMID:ATPase characteristics of myosin from nematode Caenorhabditis elegans purified by an improved method. Formation of myosin-phosphate-ADP complex and ATP-induced fluorescence enhancement. 293 26

Myosin was extracted from fresh rabbit liver using a solution with an ionic strength of 0.3 with two protease inhibitors and ATP. The myosin fraction was centrifuged at high speed in the presence of ATP and MgCl2. Its precipitate was dissolved in 0.6 M KCl, and the solution was treated with ammonium sulfate (30-60%). The fraction that precipitated was dissolved in 0.6 M KCl and put on a Sephacryl column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the myosin obtained was 90% pure. Its molecular weight was 220,000 by gel electrophoresis; by Sephacryl S-300 column chromatography, it was found to be a complex at high ionic strengths. The protein had high ATPase activity, comparable to that of the myosin prepared by D. L. Brandon (1976, Eur. J. Biochem. 65, 139-146). Electron micrographs of our myosin looked like myosins from nonmuscle cells purified by other workers. Slow preparation gave poor yields of myosin with low ATPase activity and extra bands on SDS-PAGE. Rapid handling of fresh materials is essential for obtaining myosin of satisfactory quality. Our simple method saves time and reagents.
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PMID:Simplified separation of myosin from rabbit liver. 293 10

Myosin subfragment-1 (S-1), digested with trypsin in the presence of ATP, rapidly loses its ATPase activity upon mild heat treatment even if ATP or ADP is present. The heat-treated molecule is very sensitive to further tryptic digestion. Undigested S-1 and S-1 digested in the absence of ATP are protected by nucleotides. The loss of the protective effect of nucleotides correlates with the tryptic splitting of the 25 kDa amino-terminal fragment between Arg 23 and Ile 24.
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PMID:Thermal stability of myosin subfragment-1 decreases upon tryptic digestion in the presence of nucleotides. 293 83

The purpose of this study was to determine whether a chronic swimming program could reverse the decreased cardiac function and altered myosin biochemistry found in hearts of rats with established renal hypertension. Ten wk after the onset of hypertension [midpoint (m)], hearts from normotensive controls (C) and hypertensives (H) were studied in an isolated working heart apparatus, and myosin biochemistry was analyzed. Half of the control and hypertensive animals were then subjected to a 10-wk swimming program (Sw) and their hearts were compared with those from age-matched sedentary rats. Body weight was no different at the midpoint of the study between Cm and Hm or at the end point (e) of the study among Ce, Swe, He, or H-Swe. Swimming had no effect on blood pressure in either normotensive or hypertensive rats. Dry heart weight was increased by 46% in Hm compared with Cm and by 36% in He, 21% in Swe, and 61% in H-Swe when compared with Ce. Hypertension was associated in both the mid- and end-point studies, with decreases in coronary flow, stroke work (both per gram left ventricle), ejection fraction, and midwall fractional shortening. In addition, actin-activated myosin adenosinetriphosphatase (ATPase) activity was decreased in Hm and He associated with an increase in the content of the V3 myosin isoenzyme. Although the coronary deficit was not corrected in H-Swe, stroke work, ejection fraction, and fractional midwall shortening were normalized compared with control hearts. Myosin ATPase activity and the myosin isoenzyme distribution were similarly restored in H-Swe.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Chronic swimming reverses cardiac dysfunction and myosin abnormalities in hypertensive rats. 293 53


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