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 Ca2+-dependent regulation of smooth muscle actomyosin involves a myosin light chain kinase (ATP: myosin light chain phosphotransferase). It has been shown (Dabrowska, R., Aromatorio, D., Sherry, J.M.F., and Hartshorne, D.J. 1977, Biochem. Biophys. Res. Commun. 78, 1263) that the kinase is composed of two proteins of approximate molecular weights 105 000 and 17 000. In this communication it is demonstrated that the 17 000 component is the modulator protein. This conclusion is based on: (1) the identical behavior of the 17 000 kinase component and modulator protein in assays of actomyosin Mg2+-ATPase activity, phosphorylation of myosin, and phosphodiesterase activity, and, (2) the similarity of the 17 000 kinase component and the modulator protein with respect to amino acid composition, absorption spectrum, and electrophoresis in urea-polyacrylamide gels. It is shown also that the modulator protein from smooth muscle and troponin C are distinct proteins.
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PMID:Modulator protein as a component of the myosin light chain kinase from chicken gizzard. 20

Ca2+-dependent phosphorylation of the myosin light chains in bovine aortic native actomyosin is markedly depressed in the presence of cyclic AMP and its dependent protein kinase. This inhibition occurs with either cardiac, skeletal, or aortic protein kinase plus cyclic AMP, while little or no inhibition occurs with either cyclic AMP or protein kinase alone. The extent of inhibition is related to the concentration of protein kinase and approaches a maximum of approximately 50%. Concomitant with the inhibition of myosin light chain phosphorylation is (a) an increased phosphorylation of a 100,000-dalton moiety which possibly corresponds to the myosin light chain kinase present in the native actomyosin preparation and (b) a decrease in the actomyosin Mg2+-ATPase activity. These findings suggest that modulation of actin-myosin interactions by the cAMP system directly at the level of the contractile proteins may represent a mechanism by which beta adrenergic relaxation occurs in mammalian vascular smooth muscle.
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PMID:Adenosine 3':5'-monophosphate-mediated inhibition of myosin light chain phosphorylation in bovine aortic actomyosin. 22 48

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

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

6,7-Dimethoxy-1-(3,4-dimethoxybenzyl)-4-([4-(2-methoxyphenyl)-1- piperazinyl]methyl)isoquinoline (Ro 22-4839) is a new cerebral circulation improver with vasospasmolytic properties. Preliminarily, Ro 22-4839-induced arterial relaxation was confirmed under the treatment of various constrictors and it was hardly overcome by addition of extra calcium. In this study the mode and site of action of this agent were further explored. Ro 22-4839 was found to more strongly inhibit the superprecipitation of chicken gizzard smooth muscle actomyosin (IC50 = 2.0 mumol/l) than trifluoperazine (38 mumol/l) and W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide) (220 mumol/l), an in vitro model for relaxation-contraction coupling of the smooth muscle in which calmodulin is known to play an important role through phosphorylation of myosin light chain kinase. The calmodulin antagonistic action of Ro 22-4839 was also demonstrated in other calmodulin-related reaction systems such as phosphodiesterase and hydrophobic fluorescent probe, but was very weak in Ca2+, Mg2+-ATPase of rat erythrocyte membrane. Thus, Ro 22-4839 was suggested to have a relative preference for smooth muscle contraction process unlike trifluoperazine and W-7. Moreover, Ro 22-4839 prevented the decrease in erythrocyte deformability induced by hyperosmolarity or intracellular Ca2+ accumulation, like trifluoperazine and W-7. However, Ro 22-4839 itself caused hardly an internal stomatocytic shape of erythrocytes in contrast to known calmodulin antagonists. Further, Ro 22-4839 inhibited erythrocyte membrane rupture, platelet aggregation and lipid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Calmodulin antagonistic action of the cerebral circulation improver 6,7-dimethoxy-1-(3,4-dimethoxybenzyl)-4- ([4-(2-methoxyphenyl)-1-piperazinyl]methyl)isoquinoline. 282 56

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

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

Actomyosin in smooth muscle is in a quiescent state. The mechanism or mechanisms by which Ca2+ activates the actomyosin ATPase is not clear. There is sufficient evidence for the presence of enzyme systems which phosphorylate and dephosphorylate myosin light chains. The activity of the kinase that phosphorylates the myosin is regulated by cAMP-dependent protein kinase. Phosphorylated kinase has decreased affinity for calmodulin and lower activity when compared with unphosphorylated myosin light chain kinase. The activity of myosin light chain kinase is also regulated by calcium-calmodulin. In the presence of Ca2+, myosin is phosphorylated. In the absence of Ca2+, the phosphatase activity becomes dominant; the myosin remains in the unphosphorylated form under this condition. The Mg2+-ATPase of the phosphorylated myosin is activated by actin. The maximal activation of the Mg2+-ATPase by actin requires Ca2+ and tropomyosin, a protein located on the thin filament. Hence, the actin-activation of the Mg2+-ATPase requires Ca2+ even after phosphorylation by the calcium-calmodulin dependent kinase. The regulation of actin-activated ATPase activity by myosin light chain phosphorylation is depicted in the schematic diagram. Caldesmon, an actin-binding protein which also binds to calmodulin in the presence of Ca2+, has been shown to be present in thin-filaments isolated from smooth muscle. This protein inhibits actin-activated myosin ATPase activity. The release from this inhibition requires Ca2+ and calmodulin. The possibility that caldesmon is also involved in the calcium regulation of actomyosin in smooth muscle is presently under investigation in a number of laboratories.
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PMID:Regulation of actomyosin ATPase in smooth muscle. 294 44

Fodrin, a spectrin-like actin and calmodulin binding protein, was purified to electrophoretic homogeneity from a membrane fraction of bovine brain. The effect of fodrin on smooth muscle actomyosin Mg2+-ATPase activity was examined by using a system reconstituted from skeletal muscle actin and smooth muscle myosin and regulatory proteins. The simulation of actomyosin Mg2+-ATPase by fodrin showed a biphasic dependence on fodrin concentration and on the time of actin and myosin preincubation at 30 degrees C. Maximal stimulation (50-70%) was obtained at 3 nM fodrin following 10 min of preincubation of actin and myosin. This stimulation was also dependent on the presence of tropomyosin. In the absence of myosin light chain kinase, the fodrin stimulation of Mg2+-ATPase could not be demonstrated with normal actomyosin but could be demonstrated with acto-thiophosphorylated myosin, suggesting that fodrin stimulation depends on the phosphorylation of myosin. Fodrin stimulation was shown to require the presence of both Ca2+ and calmodulin when acto-thiophosphorylated myosin was used. These observations suggest a possible functional role of fodrin in the regulation of smooth muscle contraction and demonstrate an effect on Ca2+ and calmodulin on fodrin function.
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PMID:Ca2+- and calmodulin-dependent stimulation of smooth muscle actomyosin Mg2+-ATPase by fodrin. 295 65


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