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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)

1. The transduction of energy from the oxidation of substrates by the electron transport chain or from the hydrolysis of ATP by the Mg2+-ATPase was measured in everted membrane vesicles of Escherichia coli using the energy-dependent quenching of quinacrine fluorescence and the active transport of calcium. 2. Treatment of everted membranes derived from a wild-type strain with the chaotropic agents guanidine-HC1 and urea caused a loss of energy-linked functions and an increase in the permeability of the membrane to protons, as measured by the loss of respiratory-linked proton uptake. 3. The coupling of energy to the quenching of quinacrine fluorescence and calcium transport could be restored by treatment of the membranes with N,N'-dicyclohyexylcarbodiimide. 4. Chaotrope-treated membranes were found to lack Mg2+-ATPase activity. Binding of crude soluble Mg2+-ATPase to treated membranes restored energy-linked functions. 5. Membranes prepared from a wild-type strain grown under anaerobic conditions in the presence of nitrate retained respiration-linked quenching of quinacrine fluorescence and active transport of calcium after treatment with chaotropic agents. 6. Everted membrane vesicles prepared from an Mg2+-ATPase deficient strain lacked respiratory-driven functions when the cells were grown aerobically but were not distinguishable from membranes of the wild-type when both were grown under anaerobic conditions in the presence of nitrate. 7. It is concluded (a) that chaotropic agents solubilize a portion of the Mg2+-ATPase, causing an increase in the permeability of the membrane to protons and (b) that growth under anaerobic conditions in the presence of nitrate prevents the increase in proton permeability caused by genetic or chemical removal of the catalytic portion of the Mg2+-ATPase.
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PMID:Energy transduction in Escherichia coli. The effect of chaotropic agents on energy coupling in everted membrane vesicles from aerobic and anaerobic cultures. 13 39

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

Some biochemical properties of actomyosin and myosin from elasmobranchs, Squalus acanthias and Raja tengu are compared with those of a freshwater (Cyprinus carpio) and a marine teleost (Seriola quinquiradiata). Whereas Ca2+-ATPase of teleost actomyosins are more stable in the absence of urea, the reverse is true for elasmobranchs up to 1.0 M urea. In contrast to that of teleosts, the Mg2+-ATPase of S. acanthias actomyosin shows an activation in the presence of urea, where as that of R. tengu persists. Below 1.0 M urea, there is low incorporation of DTNB into thiols of elasmobranch myosins, and losses in alpha-helicity are reversible up to 5.0 M urea. The results, thus, demonstrate that for a certain concentration of urea, elasmobranch myofibrillar proteins may exhibit a group specific tolerance to urea.
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PMID:Urea tolerance of myofibrillar proteins of two elasmobranchs: Squalus acanthias and Raja tengu. 243 54

The characteristics and specificity of inactivation of the chloroplast F1-ATPase (CF1) with 7-chloro-4-nitrobenzofurazan (Nbf-Cl) have been investigated. Inactivation of the octylglucoside-dependent Mg2+-ATPase activity of latent CF1 by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. Following inactivation of CF1 with [14C]Nbf-Cl, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the majority of the radioactive reagent incorporated is present in the beta subunit. Treatment of the enzyme with [14C]Nbf-Cl following dithiothreitol heat activation, led to similar labeling of the beta subunit and substantial incorporation of 14C into the gamma subunit. On complete inactivation, about 4 mol of Nbf-S-Cys is formed per mole of dithiothreitol-heat-activated CF1. Incorporation of 14C into the gamma subunit is prevented by prior treatment of the latent CF1 or of the dithiothreitol-heat-activated CF1 with iodoacetamide. Following incubation of the dithiothreitol-heat-activated CF1 with iodoacetamide, complete inactivation of the octylglucoside-dependent Mg2+-ATPase activity by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. After stabilization of the [14C]Nbf-O-Tyr derivative by treatment with sodium dithionite, a labeled peptide was purified. Automatic Edman degradation of this peptide revealed the sequence V-X-V-P-A-D-(D). The majority of the radioactivity was cleaved in the second cycle, the position occupied in CF1 by Tyr-beta-328, which is homologous to Tyr-beta-311, the residue reactive with Nbf-Cl in the beef heart mitochondrial F1-ATPase. When CF1, modified at Tyr-beta-328 with Nbf-Cl, is incubated at pH 9.0, the Nbf-O-Tyr adduct is hydrolyzed, leading to concomitant recovery of the ATPase activity. In double labeling experiments, two-dimensional isoelectric focusing in the presence of urea followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that 2-azido-ADP, covalently bound at the tight ADP binding site, and the tyrosine modified by [14C]Nbf-Cl are located in different beta subunits.
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PMID:Selectivity of modification when latent and activated forms of the chloroplast F1-ATPase are inactivated by 7-chloro-4-nitrobenzofurazan. 252 17

Actin in cultured bovine retinal capillary pericytes was identified and partially characterized biochemically. The filamentous actin was localized in bovine retinal capillary pericytes using a fluorescent mushroom toxin (nitrobenzoxadiazole-phallacidin) specific for actin. One-dimensional SDS-polyacrylamide-gel electrophoresis of urea-extracted proteins from bovine retinal capillary pericytes revealed a 46,000 MW protein band corresponding to an actin standard, which comprised 7.3% of the total urea-soluble proteins. Actin-activated skeletal muscle myosin Mg2+-ATPase assay, using [gamma-32P]-ATP as substrate, demonstrated functional actin in bovine retinal capillary pericyte extracts after DEAE-cellulose anion-exchange chromatography. The actin-containing protein fractions were eluted at ionic strengths between 0.25 and 0.35 M KCl. The presence of functional actin in pericytes indicated the ability to generate contractile force. This contraction-generating ability may allow pericytes to regulate microvessel caliber and to maintain the integrity of the capillary wall. A lack of this function when pericytes are preferentially lost in diabetic retinal microangiopathy could destabilize the microvessel wall and predispose the capillary to further pathologic changes.
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PMID:Actin in cultured bovine retinal capillary pericytes: morphological and functional correlation. 294 15

An investigation of the mechanism of development of hepatic encephalopathy induced by CCl4 was performed in rats. CCl4 (1.0 ml/kg three times per week for over 10 weeks) caused hepatic encephalopathy in 80% of the treated rats. Accompanying the hepatic encephalopathy were hematemesis, abdominal dropsy, and hyperammonemia, conditions observed in hepatic coma patients. The blood ammonia levels were tremendously increased in only those rats with hepatic encephalopathy. Hepatic activities of carbamylphosphate synthetase (CPS) and argininosuccinate synthetase (ASS), important enzymes of the urea cycle, were significantly inhibited by CCl4. However, the causality between the inhibition of CPS or ASS activity and the increase in blood ammonia levels was not observed. On the other hand, the content of ATP, which is a substrate of CPS and ASS, was decreased by 60% in liver of rats with hepatic encephalopathy. The activity of Mg2+-ATPase which can decompose hepatic ATP was increased by 60 and 300% in mitochondria and microsomes, respectively, of livers of rats with CCl4-induced encephalopathy. There was a good correlation between the decreased hepatic ATP content and the increased mitochondrial Mg2+-ATPase activity. Furthermore, there was also a good correlation between the increase in blood ammonia levels and the increase in Mg2+-ATPase activity in microsomes. These findings suggest that hyperammonemia, which was produced by the decrease in hepatic content and by the inhibition of CPS and ASS, may play an important role in induction of hepatic encephalopathy.
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PMID:Blood ammonia levels and hepatic encephalopathy induced by CCl4 in rats. 296 38

We have partially purified myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) from Dictyostelium discoideum. MLCK was purified 4,700-fold with a yield of approximately 1 mg from 350 g of cells. The enzyme is very acidic as suggested by its tight binding to DEAE. Dictyostelium MLCK has an apparent native molecular mass on HPLC G3000SW of approximately 30,000 D. Mg2+ is required for enzyme activity. Ca2+ inhibits activity and this inhibition is not relieved by calmodulin. cAMP or cGMP have no effect on enzyme activity. Dictyostelium MLCK is very specific for the 18,000-D light chain of Dictyostelium myosin and does not phosphorylate the light chain of several other myosins tested. Myosin purified from log-phase amebas of Dictyostelium has approximately 0.3 mol Pi/mol 18,000-D light chain as assayed by glycerol-urea gel electrophoresis. Dictyostelium MLCK can phosphorylate this myosin to a stoichiometry approaching 1 mol Pi/mol 18,000-D light chain. MLCP, which was partially purified, selectively removes phosphate from the 18,000-D light chain but not from the heavy chain of Dictyostelium myosin. Phosphatase-treated Dictyostelium myosin has less than or equal to 0.01 mol Pi/mol 18,000-D light chain. Phosphatase-treated myosin could be rephosphorylated to greater than or equal to 0.96 mol Pi/mol 18,000-D light chain by incubation with MLCK and ATP. We found myosin thick filament assembly to be independent of the extent of 18,000-D light-chain phosphorylation when measured as a function of ionic strength. However, actin-activated Mg2+-ATPase activity of Dictyostelium myosin was found to be directly related to the extent of phosphorylation of the 18,000-D light chain. MLCK-treated myosin moved in an in vitro motility assay (Sheetz, M. P., and J. A. Spudich, 1983, Nature (Lond.), 305:31-35) at approximately 1.4 micron/s whereas phosphatase-treated myosin moved only slowly or not at all. The effects of phosphatase treatment on the movement were fully reversed by subsequent treatment with MLCK.
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PMID:Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function. 303 87

The reactivity of the sulfhydryl groups in colonic myosin B to N-ethylmaleimide (NEM) was studied under various conditions. A higher concentration of NEM was required to alter the ATPase activity as compared with skeletal myosin B. A chemical modification of colonic myosin B at a low ionic strength brought about the marked inhibition of EDTA-ATPase and 30-40% activation of Mg2+-ATPase, but had little effect on Ca2+-ATPase. The same results were obtained with NEM modification at a high ionic strength. The presence of 10 mM MgCl2 in the reaction medium for NEM modification caused the activation of Ca2+-ATPase and remarkable inhibition of EDTA-ATPase, suggesting that SH1 is here reactive to NEM. NEM modification of colonic myosin B in the presence of ATP resulted in the inhibition of not only EDTA-ATPase but also Ca2+- and Mg2+-ATPases although the degree of inhibition was different. Addition of 2.0 M urea to the modification medium caused the simultaneous inhibition of EDTA- and Ca2+-ATPase but little change in Mg2+-ATPase. Based on these results, the presence of three specific sulfhydryl groups, SHa, SH1, and SH2, is discussed.
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PMID:The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. VII. A chemical modification of colonic smooth myosin B with N-ethylmaleimide. 611 53

alpha-Actinin purified from chicken gizzard smooth muscle was characterized in comparison with alpha-actinins from chicken striated muscles, or fast-skeletal muscle, slow-skeletal muscle, and cardiac muscle. The gizzard alpha-actinin molecule consisted of two apparently identical subunits with a molecular weight of 100,000 on SDS-polyacrylamide gel electrophoresis, as do striated-muscle alpha-actinins. Its isoelectric points in the presence of urea were similar to the striated-muscle counterparts. Despite these similarities, distinctive amino acid sequences between smooth-muscle alpha-actinin and striated-muscle alpha-actinins were revealed by peptide mapping using limited proteolysis in SDS. Gizzard alpha-actinin was immunologically distinguished from striated-muscle alpha-actinins. Gizzard alpha-actinin formed bundles of gizzard F-actin as well as of skeletal-muscle F-actin, but could not form any cross-bridges between adjacent actin filaments under conditions where skeletal-muscle alpha-actinin could. Temperature-dependent competition between gizzard alpha-actinin and tropomyosin on binding to gizzard thin filaments was demonstrated by electron microscopic observations. Gizzard alpha-actinin promoted Mg2+-ATPase activity of reconstituted skeletal actomyosin, gizzard acto-skeletal myosin, and gizzard actomyosin. This promoting effect was depressed by the addition of gizzard tropomyosin. These findings imply that, despite structural differences between gizzard and striated-muscle alpha-actinin molecules, they function similarly in vitro, and that gizzard alpha-actinin can interact not only with smooth-muscle actin (gamma- and beta-actin) but also with skeletal-muscle actin (alpha-actin).
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PMID:Molecular properties and functions in vitro of chicken smooth-muscle alpha-actinin in comparison with those of striated-muscle alpha-actinins. 621 60

Actomyosin complex was extracted from the brain cortex in a medium consisting of low salt, ATP, and EDTA, in the presence of protease inhibitors, followed by ammonium sulfate fractionation. Myosin was then purified from the actomyosin. Myosin obtained according to the procedure used was significantly contaminated with actin high (greater than 200,000 dalton) and low molecular weight proteins. Therefore, an alternative method based on affinity chromatography (Blue Dextran/Sepharose) and gel filtration (Sepharose 4B) was developed to purify myosin. This procedure yielded myosin that was greater than 95% pure as judged by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit composition of purified brain myosin was monitored by sodium dodecyl sulfate-polyacrylamide gel also containing a urea gradient. A closely migrating triplet in the heavy chain and three light chains, LC1, LC2, and LC3, of Mr 21,000, 19,000, and 17,000, respectively, were observed. These findings raise the possibility of the existence of myosin isoenzymes in the brain. Brain myosin formed bipolar thick filaments in 0.075 M KCl and MgCl2. At low ionic strength, the Mg2+-ATPase activity of myosin was stimulated 3- to 3.5-fold in the presence of skeletal muscle f-actin. Brain myosin also hydrolyzed other nucleotides; the rate of hydrolysis was ITP greater than ATP approximately equal to CTP greater than GTP approximately equal to UTP. The substrate (ATP) saturation curve in the presence of 10 mM CaCl2 and 0.6 M KCl was complex and consisted of plateau regions. The Arrhenius plot of the Ca-ATPase data was linear, whereas with ITPase, it was biphasic with a break occurring around 20 degrees C.
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PMID:Purification and characterization of myosin from calf brain. 622 62


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