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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 contractile proteins actin and myosin have been isolated from the soluble phase of guinea-pig polymorphonuclear leucocytes and partially characterised. Two forms of actin have been identified, designated 'Mg-actin' and 'KCl-actin'. They have different polymerising properties but their propensity to form synthetic homologous and heterologous actomyosins and to inhibit DNAase-1 does not significantly differ. Both show beta and gamma isoelectric forms in focusing gels and the Mg-actin accounts for about 5% of the soluble-phase protein and te KCl-actin around 2%. Leucocyte myosin has been isolated by affinity chromatography on N6-ADP-Sepharose with a good enrichment of both Ca2+-ATPase and the ATPase activity measured in the absence of Ca2+ or Mg2+ and in the presence of EDTA. This protein, too, has the capacity to form synthetic homologous and hybrid actomyosins with enhancement of the basal Mg2+-ATPase activity. The ratio of actin to myosin in the leucocyte calculated on a molar basis is well in excess of 100, a figure consistent with the findings from other non-muscle cells.
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PMID:The isolation and characterisation of guinea-pig polymorphonuclear leucocyte actin and myosin. 610 49

The properties of Mg2+-ATPase in the vacuole of Saccharomyces cerevisiae were studied, using purified intact vacuoles and right-side-out vacuolar membrane vesicles prepared by the method of Y. Ohsumi and Y. Anraku ((1981) J. Biol. Chem. 256, 2079). The enzyme requires Mg2+ ion but not Ca2+ in. Cu2+ and Zn2+ ions inhibit the activity. The optimal pH is at pH 7.0. The enzyme hydrolyzes ATP, GTP, UTP, and CTP in this order and the Km value for ATP was determined as 0.2 mM. It does not hydrolyze ADP, adenosyl-5'-yl imidodiphosphate, or p-nitrophenyl phosphate. ADP does not inhibit hydrolysis of ATP by the enzyme. The activities of intact vacuoles and of vacuolar membrane vesicles were stimulated 3- and 1.5-fold, respectively, by the protonophore uncoupler 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile and the K+/H+ antiporter ionophore nigericin. Sodium azide at a concentration exerting an uncoupler effect also stimulated the activity. The activity was sensitive to the ATPase inhibitor N,N'-dicyclohexylcarbodiimide, but not to sodium vanadate. The ATP-dependent formation of an electrochemical potential difference of protons, measured by the flow-dialysis method, was determined as 180 mV, with contribution of 1.7 pH units, interior acid, and of a membrane potential of 75 mV. It is concluded that the Mg2+-ATPase of vacuoles is a new marker enzyme for these organelles and is a N,N'-dicyclohexylcarbodiimide-sensitive, H+-translocating ATPase whose catalytic site is exposed to the cytoplasm.
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PMID:Properties of H+-translocating adenosine triphosphatase in vacuolar membranes of SAccharomyces cerevisiae. 611 10

The kinetics of the hydrogen-deuterium exchange reaction in sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle was followed by infrared absorption measurements. The exchange rate in SR was much lower than that in the soluble proteins reported so far. When adenylyl-imidophosphate (AMPPNP, an TP analog) was present, the exchange rate was lower than that in free SR and it was the lowest when ADP was present. The effect of the nucleotides on the exchange rate reflects the conformational change of the Ca2+, Mg2+-ATpase of SR membranes on binding the nucleotides. The structure of e Ca2+, Mg2+-ATPase is more restricted in the following order: SR + ADP greater than SR + AMPPNP greater than free SR.
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PMID:Effects of ADP and AMPPNP on the hydrogen-deuterium exchange kinetics in Ca2+, Mg2+-ATpase of sarcoplasmic reticulum. 611 50

We have identified an anion-sensitive Mg2+-ATPase in adenohypophyseal secretory granule membranes. This enzyme is unaffected by sodium, ouabain, and calcium. By electron microscopic morphology, sedimentation properties, nucleotide substrate utilization, and marker enzyme studies, this activity is clearly shown to be intrinsic to the granule membranes. The kinetics for ATP saturation were complex, as curvilinear Lineweaver-Burk plots were obtained with 2 mM magnesium. However, an approach to linearity was obtained (Km for ATP, approximately 0.27 mM) with low concentrations of free magnesium. Many anions and anion-transport blockers significantly influenced enzyme activity. Stimulatory anions in decreasing order of potency were bisulfite greater than sulfite greater than isethionate greater than bicarbonate; Ka values were 2.5 mM for sulfite and 10.8 mM for bicarbonate. Acetate, borate, chloride, citrate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, nitrite, oxalate, 1,3-piperazinediethanesulfonic acid, and sulfate were without major effect. Inhibitory anions in decreasing potency order were azide greater than thiocyanate greater than fluoride greater than nitrate. Anionic stimulation of the granule membrane Mg2+-ATPase linearized the Lineweaver-Burk plots by shifting the enzyme to its higher Km state. In addition, sulfite competitively reversed the produce inhibition exerted by ADP. Anion transport-blockers inhibited the enzyme; of those tested, the most potent was 4-acetamido-4-isothiocyano-stilbene-2,2'-disulfonic acid, with a Ki of 0.17 mM; pyridoxal phosphate, sulfisoxazole, and ethacrynic acid also inhibited enzyme activity. The protein-binding dye p-sulfobenzene-azo-o-sulfobenzene-azo-beta-naphthol-3,6-disulfonic acid, structurally similar to transport blockers, was a potent inhibitor, with a Ki of 2.8 mM. These data on pituitary secretory granule ATPase raise the possibility that the granule membranes may function in anion or proton transport, perhaps in relation to exocytosis and hormone secretion.
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PMID:Identification and characterization of an anion-sensitive Mg2+-ATPase in pituitary secretory granule membranes. 611 65

Interaction of adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) with Ca2+,Mg2+-ATPase of sarcoplasmic reticulum was studied. The nucleotide was slowly hydrolyzed by the ATPase at 30 degrees C at a rate of about 0.5% that of ATP hydrolysis. Whereas at 0 degrees C, ATP gamma S showed only a limited reactivity toward the ATPase in that a thiophosphorylated intermediate was formed and ADP was released, but hydrolysis of the intermediate to complete the catalytic cycle did not occur. A fairly stable analog of the E-P intermediate could thus be obtained. Presence of the thiophosphorylated intermediate was indicated by the [3H]ADP in equilibrium ATP gamma S exchange reaction and also by using [35S]ATP gamma S. When the ATPase was reacted with ATP gamma S at 0 degrees C in the presence of ferricyanide, EP-forming activity was rapidly lost. Free Ca2+ ions were required for this inactivation. Disulfide bond formation between a cysteinyl residue located near the substrate binding site and the enzyme-bound ATP gamma S or the thiophosphorylated intermediate was suggested by the fact that 2-mercaptoethanol reversed the inactivation. The reaction may prove to be a useful tool for affinity labeling of the active site of the ATPase.
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PMID:Interaction of adenosine-5'-O-(3-thiotriphosphate) with Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum. 612 38

Mechanistic studies of Ca2+ transport by the Ca2+-Mg2+-ATPase of skeletal sarcoplasmic reticulum are reviewed, and a unifying model is proposed. The significant steps in the transport cycle are modeled in terms of occupation and disposition of three binding sites on the enzyme: a) two translocation sites capable of binding to Ca2+ or a charge-stoichiometric amount of alkali cation (M+) or H+, b) an ATP-ADP-binding site, and c) a phosphorylation or phosphate-binding site. The normal transport cycle is characterized as the following sequence of steps: a) binding of two Ca2+ and Mg-ATP to external sites with high affinity and random order, b) enzyme phosphorylation, c) inward translocation of the Ca2+-laden sites, d) Ca2+ release to the sarcoplasmic reticulum lumen and ADP release to the external medium (random order), e) binding of Mg2+ or a charge-stoichiometric amount of K+ plus H+ to the translocators, f) dephosphorylation, g) the return of the K+- and H+-laden translocators to the outside, and h) dissociation of K+ and H+ from the translocator and completion of the cycle with step a. The enzyme is characterized as a Ca2+-K+ plus H+ countertransporter. The K+ plus H+ remove Ca2+ from the inwardly oriented translocator, thereby relieving a product inhibition and increasing the rate of enzyme dephosphorylation.
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PMID:Mechanism of Ca2+ transport by Ca2+-Mg2+-ATPase pump: analysis of major states and pathways. 612 4

Various reaction intermediates of sarcoplasmic reticulum Ca2+,Mg2+-ATPase were stabilized and accumulated by modifying a specific SH group or by using nucleotide analogs. Conformational changes of the Ca2+,Mg2+-ATPase during the catalytic cycle were studied in the stabilized intermediates by the use of fluorescent and spin probes, which were introduced at specific SH groups of ATPase, namely one highly reactive but functionally nonessential (SHN) and one essential for the decomposition of the E-P intermediate (SHD) [Kawakita, M., et al. (1980) J. Biochem. 87, 609-617]. The fluorescence intensity of N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide attached to SHD decreased by 2.5% upon addition of 10 microM AMP-P(NH)P provided that Ca2+ was also present. The AMP-P(NH)P-induced fluorescence change could also be detected by using other fluorescent probes such as N-[p-(2-benzimidazolyl)phenyl]maleimide and N-(1-anilinonaphthyl-4)maleimide. Moreover, labeling at SHN gave similar results. When SHN was labeled with N-[p-(2-benzimidazolyl)phenyl]maleimide, the fluorescence intensity also decreased by 2.5% upon addition of ATP only in the presence of Ca2+, where E-P formation took place. A conformational difference between ECa1-P X ADP and ECa1-P was suggested from saturation transfer ESR measurement of spin-labeled ATPase by using ADP beta S as an ADP analog to cause accumulation of ECa1-P X ADP beta S complex. Possible structural similarities among some of the intermediates are discussed based on these findings.
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PMID:Studies on conformational transitions of Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum. II. Conformational characteristics of stabilized reaction intermediates as revealed by fluorescent and paramagnetic probes. 613 71

Placental polypeptides present in crude preparations of transforming growth factors stimulate glycolysis when added to quiescent 3T3 cells, normal rat kidney, and chick embryo fibroblasts. The stimulation was apparent over a time period of at least 90 min and was seen at glucose concentrations ranging from 1 to 30 mM. Duramycin, an antibiotic isolated from Streptomyces cinnamomeus, inhibits the polypeptide-stimulated and nonstimulated glycolysis of intact cells, since it permeabilizes cells to Pi and nucleotides. However, duramycin also inhibits the Na+-K+-ATPase as well as the ouabain-insensitive Mg2+-ATPase of plasma membranes. Duramycin has no effect on glycolysis catalyzed by cell-free extracts of Ehrlich ascites tumor cells in the presence of mitochondrial ATPase but partially inhibits glycolysis when ADP and Pi are generated by ATPases of plasma membrane preparations.
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PMID:Stimulation of glycolysis by placental polypeptides and inhibition by duramycin. 614 64

A FORTRAN computer program capable of calculating the steady-state behavior of the Ca2+ -Mg2+-ATPase pump of skeletal sarcoplasmic reticulum under all conditions of reactant and product concentrations is described. The model describes the behavior of the enzyme in terms of occupation of three binding sites: (a) a translocator site which can bind Ca2+, K+, H+, or Mg2+, (b) an ATP/ADP binding site, and (c) a phosphorylation and phosphate binding site. The translocator site can move across the membrane in the Ca2+-laden and K+ + H+-laden form, thereby accomplishing Ca2+ for K+ + H+ countertransport. The rate constants for ion binding and the translocation reactions vary as a function of translocator orientation, ATP and ADP occupancy, phosphorylation, and phosphate binding. Rate constants for the binding and the reactant and product concentrations and association reactions and other transformations between states of the enzyme are entered and the computer program solves for the steady-state concentrations of all states of the enzyme and for the turnover number of the enzyme. The program contains a matrix of differential equations for creation and destruction of all states of the enzyme using the steady-state assumption together with the rule of conservation of the total enzyme. The matrix of equations and states is solved by Gaussian elimination. The program presents the distribution of enzyme states in histogram fashion and is capable of presenting the concentration of a particular state or the rate of turnover as a function of any of the reactant or product concentrations. Three demonstrations of the utility of the program and predictive power of the model are given.
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PMID:Modeling the steady-state behavior of the Ca2+ -Mg2+ -ATPase pump of sarcoplasmic reticulum. 614 52

Interaction between Gd3+ and Tb3+ ions and Ca2+,Mg2+-ATPase of sarcoplasmic reticulum was studied. Three classes of lanthanide-ion binding sites with different affinities were distinguished. Binding of Gd3+ to the site with the highest affinity seemed to occur at less than 10(-6)M free Gd3+ and resulted in severe inhibition of ATPase activity. The reaction rates of both E-P formation and decomposition in the forward direction were inhibited in parallel with this binding, whereas ADP-dependent decay of E-P in the backward direction was not. At these Gd3+ concentrations, Ca2+-binding to the transport site was not inhibited. Binding of Gd3+ and Tb3+ to the Ca2+-transport site did occur, but more than 10(-5)M free Gd3+ or Tb3+ was required for effective competition with Ca2+ for that site. Gd3+ bound to the transport site in place of Ca2+ did not activate the E-P intermediate formation. Addition of 10(-1)M Tb3+ to a suspension of sarcoplasmic reticulum membranes resulted in marked enhancement of Tb3+ fluorescence, which is due to an energy transfer from aromatic amino acid residues of ATPase to Tb3+ ions bound to the low affinity site of the enzyme. Gd3+ and Mn2+ competed with Tb3+ for that site, but Ca2+, Zn2+, and Cd2+ did not.
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PMID:Characterization of Gd3+ and Tb3+ binding sites on Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum. 614 74


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