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:3.6.3.1 (Mg2+-ATPase)
1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 20,000-dalton light chain of bovine platelet myosin is phosphorylated at two sites by myosin light chain kinase. The first and second phosphorylation sites are at a serine and a threonine residue, respectively. The location of the phosphorylation sites was determined by using limited proteolysis. The N-terminal sequence of the 17,000-dalton tryptic fragment of platelet myosin 20,000-dalton light chain was found to be identical with that of gizzard 20,000-dalton light chain from Ala-17 to Phe-33. On the basis of these results and the distribution of 32P among the proteolytic fragments, it was concluded that serine-19 and threonine-18 were the two phosphorylation sites. Phosphorylation at the threonine residue markedly increases the actin-activated ATPase activity of myosin. It was found that platelet myosin forms 10S and 6S conformations and its Mg2+-ATPase activity parallels the transition from the 6S to the 10S conformation. The conformational transition was influenced by phosphorylation at both sites, and the phosphorylation at the threonine residue further shifted the equilibrium toward the 6S conformation. The phosphorylation at the threonine residue also induced thick filament formation in the presence of ATP. These results suggest that the phosphorylation at the threonine residue as well as at the serine residue may play an important role in the contractility of nonmuscle cells.
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PMID:Phosphorylation of a second site for myosin light chain kinase on platelet myosin. 253 45

Myosin was purified from bovine erythrocytes by chromatography on DEAE-cellulose, Sepharose CL-4B, hydroxylapatite, and DEAE-5PW. The yield was about 200 micrograms/L of packed cells. From SDS-polyacrylamide gels, the purity was estimated to be greater than 95%. The bovine erythrocyte myosin is composed of heavy chains of 200 kDa and light chains of 20 and 17 kDa, in a molar stoichiometry of 1. Myosin was also purified from human erythrocytes by the same method. The molecular weights of two light chains were 26K and 19.5K which confirmed the earlier reports [Fowler, V. M., Davis, J. Q., & Bennet, V. (1985) J. Cell Biol. 100, 47-55; Wong, A. J., Kiehart, D. P., & Pollard, T.D. (1985) J. Biol. Chem. 260, 46-49]. Phosphorylation by gizzard myosin light chain kinase, to a level of 1 mol of phosphate/mol of 20-kDa light chain, increased actin-activated ATPase, and the extent of activation was dependent on the MgCl2 concentration. Both Ca2+-ATPase and Mg2+-ATPase activities were dependent on KCl concentration and markedly decreased below 0.3 M KCl. Mg2+-ATPase of phosphorylated myosin, while more resistant to decreasing ionic strength, was also decreased below 0.2 M KCl. These results are similar to those obtained with smooth muscle myosin and suggest that the 10S-6S transition occurs. In confirmation of this, gel filtration, viscosity, and electron microscopy (rotary shadowing) show that erythrocyte myosin forms extended and folded conformations in high and low salt, respectively. It is proposed that each conformation is characterized by distinct enzymatic properties.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Correlation of enzymatic properties and conformation of bovine erythrocyte myosin. 254 59

Several techniques were used to investigate the possibility that smooth muscle tropomyosin interacts with smooth muscle myosin. These experiments were carried out in the absence of actin. The Mg2+-ATPase activity of myosin was activated by tropomyosin. This was most marked at low ionic strength but also occurred at higher ionic strength with monomeric myosin. For myosin and HMM, the activation of Mg2+-ATPase by tropomyosin was greater at low levels of phosphorylation. There was no detectable effect of tropomyosin on the Mg2+-ATPase activity of S1. The KCl dependence of myosin viscosity was influenced by tropomyosin, and in the presence of tropomyosin, the 6S to 10S transition occurred at lower KCl concentrations. From the viscosity change, an approximate stoichiometry of 1:1 tropomyosin to myosin was estimated. The phosphorylation dependence of viscosity, which reflects the 10S-6S transition, also was altered in the presence of tropomyosin. An interaction between myosin and tropomyosin was detected by fluorescence measurements using tropomyosin labeled with dansyl chloride. These results indicate that an interaction occurs between myosin and tropomyosin. In general, the interaction is favored at low ionic strength and at low levels of phosphorylation. This interaction is not expected to be competitive with the formation of the actin-tropomyosin complex, but the possibility is raised that a direct interaction between myosin and tropomyosin bound to the thin filament could modify contractile properties in smooth muscle.
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PMID:Interaction of smooth muscle tropomyosin and smooth muscle myosin. Effect on the properties of myosin. 254 78

An antibody obtained by immunizing a rabbit with purified bovine brain myosin was found to react with the tail portion of the myosin heavy chain. An Fab fragment obtained by limited papain digestion of the antibody was allowed to bind to brain myosin, and the complex of the Fab fragment and brain myosin (Fab-myosin) was isolated. On examination of the rotary-shadowed Fab-myosin by electron microscopy, most of the Fab fragment was located on the middle to C-terminal regions of the tails of the myosin molecules. The solubility of Fab-myosin in low salt solutions was higher than that of control brain myosin. Fab-myosin was found to form small irregular aggregates in low salt solutions instead of regular bipolar filaments, and the relative population of the monomeric form of myosin molecules observed for the Fab-myosin was much larger than that observed for the control myosin. The actin-activated Mg2+-ATPase activity of Fab-myosin was stimulated two- to threefold by phosphorylation of the light chains with myosin light chain kinase, as observed for the control brain myosin. Furthermore, the levels of the ATPase activity of the phosphorylated and dephosphorylated Fab-myosins were similar to those of the phosphorylated and dephosphorylated control myosins, respectively. The superprecipitation activity of Fab-myosin was also highly dependent on phosphorylation of the light chains. Although control brain myosin formed a large superprecipitate network which contracted to a dense particle, Fab-myosin generated only numerous tiny superprecipitates under the same conditions. From these results it was deduced that a regular filamentous state of brain myosin was not prerequisite for its actin-activated Mg2+-ATPase and superprecipitation activities but was indispensable for the formation of a large and well contractible superprecipitate.
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PMID:Physical, enzymatic, and contractile properties of brain myosin with anti-brain myosin Fab fragment bound on its tail. 275 76

Caldesmon is a major calmodulin- and actin-binding protein of smooth muscle which interacts with calmodulin in a Ca2+-dependent manner or with actin in a Ca2+-independent manner. Isolated caldesmon is capable of inhibiting the actin-activated Mg2+-ATPase of smooth-muscle myosin, suggesting a possible physiological role for caldesmon in regulating the contractile state of smooth-muscle. Caldesmon can be phosphorylated in vitro by a co-purifying Ca2+/calmodulin-dependent protein kinase and dephosphorylated by a protein phosphatase, both of which are present in smooth muscle. We investigated further the phosphorylation of caldesmon and the effects which phosphorylation has on the functional properties of the protein. The kinetics of caldesmon phosphorylation were similar whether the caldesmon substrate was free or bound to actin, actin/tropomyosin or thin filaments. Caldesmon containing endogenous kinase activity was rapidly phosphorylated (to approx. 1 mol of Pi/mol of caldesmon in 5 min) when reconstituted with actin, myosin, tropomyosin, calmodulin and myosin light-chain kinase in the presence of Ca2+ and MgATP2-. Under conditions in which unphosphorylated caldesmon showed substantial inhibition of the actin-activated myosin Mg2+-ATPase, no inhibition was observed with phosphorylated caldesmon. This was the case whether caldesmon was phosphorylated before addition to the actomyosin Mg2+-ATPase system, or phosphorylation was allowed to take place during the ATPase reaction. Binding studies revealed maximal binding of 1 mol of unphosphorylated caldesmon/9.5 mol of actin and 1 mol of phosphorylated caldesmon/11.7 mol of actin. All the bound phosphorylated caldesmon could be released by Ca2+/calmodulin, with half-maximal release at 0.11 microM-Ca2+, whereas only 62% of the bound unphosphorylated caldesmon could be removed, with half-maximal release at 0.16 microM-Ca2+. However, under conditions in which inhibition of actomyosin Mg2+-ATPase activity by non-phosphorylated but not by phosphorylated caldesmon was observed, both forms of caldesmon would remain bound to the thin filament. These observations suggest a possible mechanism whereby caldesmon phosphorylation may prevent its inhibitory action on the actomyosin Mg2+-ATPase.
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PMID:The effects of phosphorylation of smooth-muscle caldesmon. 282 3

We have examined the effects on the activities of three calmodulin-dependent enzymes (cAMP phosphodiesterase, caldesmon kinase and myosin light chain kinase) of the dihydropyridine Ca2+ channel blocker felodipine and three analogues (p-chloro, oxidized and t-butyl) exhibiting different pharmacological potencies. The cAMP phosphodiesterase was inhibited completely by felodipine and the p-chloro analogue with IC50 values of 3.7 and 1.5 microM respectively. The oxidized and t-butyl analogues were relatively ineffective in inhibiting cAMP phosphodiesterase. Felodipine and the p-chloro analogue inhibited the basal (Ca2+/calmodulin-independent) activity of cAMP phosphodiesterase as well as the calmodulin-stimulated activity. Calmodulin was relatively ineffective in preventing inhibition of cAMP phosphodiesterase by felodipine and the p-chloro analogue. These observations suggest that felodipine may act directly on the phosphodiesterase as well as through calmodulin. Felodipine and the p-chloro analogue inhibited Ca2+/calmodulin-dependent caldesmon kinase with similar potencies (IC50 = 17.4 microM), whereas the oxidized and t-butyl analogues caused no inhibition. Similarly, felodipine and the p-chloro analogue inhibited myosin light chain kinase activity whether the isolated 20 kD light chain (IC50 = 12.6 microM) or intact myosin (IC50 = 11.0 microM) was used as substrate. Inhibition in each case was prevented by excess calmodulin. The oxidized and t-butyl derivatives caused little or no inhibition. Finally, the effects of felodipine and the three analogues on two processes which are dependent on myosin phosphorylation were examined, namely the actin-activated Mg2+-ATPase activity of myosin and the assembly of myosin filaments. Felodipine and the p-chloro analogue inhibited the actin-activated Mg2+-ATPase activity of smooth muscle myosin (IC50 = 25.1 microM). The oxidized and t-butyl analogues exhibited no inhibition. Similarly, felodipine and the p-chloro analogue blocked myosin filament assembly induced by low concentrations of calmodulin, whereas the oxidized and t-butyl analogues did not. Again, inhibition of the actin-activated myosin Mg2+-ATPase and myosin filament assembly by felodipine and the p-chloro analogue could be reversed by raising the calmodulin concentration. These observations suggest that some of the pharmacological actions of felodipine on smooth muscle may involve inhibition of calmodulin-dependent enzymes which are functionally involved in the regulation of smooth muscle contraction.
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PMID:Effects of felodipine (a dihydropyridine calcium channel blocker) and analogues on calmodulin-dependent enzymes. 283 1

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

Caldesmon, an actin- and calmodulin-binding protein of smooth muscle, is a protein serine/threonine kinase capable of Ca2+/calmodulin-dependent autophosphorylation [Scott-Woo & Walsh (1988) Biochem. J. 252, 463-472]. Phosphorylation nullifies the inhibitory effect of caldesmon on the actin-activated Mg2+-ATPase activity of smooth-muscle myosin [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have characterized the kinase activity of caldesmon of chicken gizzard smooth muscle. Autophosphorylation requires Ca2+/calmodulin, but is unaffected by other second messengers (Ca2+/phospholipid/diacylglycerol, cyclic AMP or cyclic GMP), and is inhibited by the calmodulin antagonists chlorpromazine and compound 48/80, with 50% inhibition at 39.8 microM and 12.0 ng/ml respectively. Half-maximal activation of autophosphorylation occurs at 60-80 nM-Ca2+ and 0.14 microM-calmodulin, and maximal activity at 0.14-0.18 microM-Ca2+ and 1 microM-calmodulin; activation is gradually lost at higher Ca2+ and calmodulin concentrations. Autophosphorylation is pH-dependent, with maximal activity over the range pH 7-9, and requires free Mg2+ in addition to the MgATP2- substrate. The Km for ATP is 15.6 +/- 4.1 microM (mean +/- S.D., n = 4), and kinase activity is inhibited by increasing ionic strength [half-maximal inhibition at I = 0.094 +/- 0.009 M (mean +/- S.D., n = 4)]. Autophosphorylation does not affect the rate of hydrolysis of caldesmon (free or bound to calmodulin) by alpha-chymotrypsin. However, a slight difference in peptides generated from phospho- and dephospho-forms of caldesmon is observed. The binding of phospho- or dephospho-caldesmon to F-actin protects the protein against chymotryptic digestion, but does not alter the pattern of peptide generation. Characterization of proteolytic fragments of caldesmon generated by alpha-chymotrypsin and Staphylococcus aureus V8 protease enables localization of the phosphorylation sites and the kinase active site within the caldesmon molecule.
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PMID:Characterization of the autophosphorylation of chicken gizzard caldesmon. 285 Jul 99

1. The myosin molecule from Ehrlich ascites tumour cells consists of heavy chains of about 200 kDa and three species of light chains of 20, 19 and 15 kDa. 2. The heavy chain can be phosphorylated in vitro either by endogenous Ca2+-independent kinase or by casein kinase II. 3. The 20 and 19 kDa light chains can be phosphorylated either by an endogenous kinase or by myosin light chain kinase from chicken gizzard. 4. The Ca2+-ATPase activity of the purified myosin was 0.3 mumol/min mg protein. The Mg2+-ATPase activity was activated 14-fold by actin upon the light chain phosphorylation.
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PMID:Purification of myosin from Ehrlich ascites tumour cells (phosphorylation of its light chain and heavy chain). 285 95

By peptide isolation and analysis, it has been shown that the dansyl fluorophore of dansylcadaverine [N-(5-aminopentyl)-5-(dimethylamino)naphthalene-1-sulfonamide] transfers to Gln-41 of actin from rabbit skeletal muscle when the reaction is catalyzed by guinea pig liver transglutaminase. As a function of time, the degree of labeling asymptotically approaches 1 mol of dansyl/l mol of actin. About 80-85% of the attached dansyl fluorophore was found at Gln-41. Such labeled G-actin polymerizes to the same extent as control actin, but the polymerization rate is greater and the critical concentration is less than for control actin. Complete polymerization is accompanied by a 1.5-2.0-fold increase in the emission intensity of the attached fluorophore. Labeled F-actin thus obtained activates myosin subfragment 1 (S-1) Mg2+-ATPase activity with the same Kapp, and to the same Vmax, as control actin; moreover, when such labeled F-actin is cross-linked to S-1 by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, the resulting superactivation of Mg2+-ATPase is the same as that attained with control actin. The attributes of this label thus make it an ideal reporter of events in the N-terminal 10-kilodalton region of actin, and a new topological point for proximity mapping.
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PMID:A novel actin label: a fluorescent probe at glutamine-41 and its consequences. 289 15


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