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)

phiX174 DNA-dependent DNA synthesis is catalyzed in vitro by the combination of at least 11 purified protein fractions: dnaB, dnaC(D), and dnaG gene products, DNA polymerase III, DNA elongation factors I and II, DNA binding protein, and replication factors W, X, Y, and Z. The reaction requires ATP, 4 dNTPs, and Mg+2 and is specific for phiX174 (or phiXahb) DNA. Purified replication factor Y contains phiX174 (or phiXahb) DNA-dependent ATPase (or dATPase) activity. The ATPase activity is poorly stimulated by other single-stranded DNA, by double-stranded DNA, or by RNA. The products of the phiX174 DNA-dependent ATPase activity of factor Y are Pi and ADP (or dADP). The association of phiX174 DNA-dependent ATPase activity with factor Y was shown in the following ways: (a) the two activities copurified with a constant ratio; (b) they comigrated on native polyacrylamide gel electrophoresis; (c) both activities were heat-inactivated at the same rate; and (d) both showed identical patterns of N-ethylmaleimide sensitivity.
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PMID:Association of phiX174 DNA-dependent ATPase activity with an Escherichia coli protein, replication factor Y, required for in vitro synthesis of phiX174 DNA. 12 75

ATP, added to the solution bathing the nutrient (serosal) surface of isolated frog gastric mucosa, was found to break down rapidly to ADP, inorganic phosphate and other products. This activity is due to an ectoenzyme, i.e. to an enzyme system easily accessible to the bathing solution. This conclusion follows from experiments which showed that penetration of ATP into the mucosal cells occurred at a much slower rate: leakage of inorganic phosphate and adenine nucleotides from mucosal cells was also minor. The surface ATPase may reflect the operation of a mechanism at the nutrient surface involved in acid secretion.
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PMID:Hydrolysis of exogenous ATP by isolated frog gastric mucosa. 12 22

The rates of the ATPase [EC 3.6.1.3] reaction of the H-meromyosin-F-actin-relaxing protein system were measured in 2 mM MgCl2, 50mM KC1, and 10mM Tris-HC1 at pH 7.8 and 20 degrees in the presence and absence of 0.05-0.1 mM Ca2+ ions. The concentrations of H-meromyosin (HMM) and the F-actin-relaxing protein (F-A-PR) complex were 3.4 and 3 mg/ml, respectively, and the ATPase reaction was coupled with 4 mg/ml of pyruvate kinase [EC 2.7.1.40] and 1 or 20 mM phosphoenolpyruvate to regenerate ATP. The amount of ADP bound to HMM during the ATPase reaction was determined by measuring the amount of ADP remaining in the reaction mixture. The amount of ATP bound to HMM was determined by subtracting the amount of bound ADP from the total amount of nucleotides bound to HMM, which was measured by a rapid flow-dialysis method. The following results were obtained. 1. The ATPase activity of the HMM-F-A-RP system increased linearly with increase in the amount of ATP added, and was independent of the presence of 0.05 mM Ca2+, when the amount of ATP added was less than 1 mole/mole of HMM. In the presence of 0.05 mM Ca2+, the ATPase activity reached a maximal level when 1.2-1.5 mole of ATP was added per mole of HMM, and maintained this level even at 3 moles of added ATP/mole of HMM. In the presence of 3mM EGTA, the ATPase activity decreased with increase in the amount of ATP added, from 1.5 to 3 moles of ATP/mole of HMM, and reached the level of the HMM ATPase reaction at 3 moles of added ATP/mole of HMM. Similar results were observed when the concentration of HMM was maintained at 3.4 mg/ml and the concentration of the F-A-RP complex was decreased from 3 to 1 or 0.5 mg/ml.
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PMID:The amounts of adenosine di- and triphosphates bound to H-meromyosin and the adenosinetriphosphatase activity of the H-meromyosin-F-actin-relaxing protein system in the presence and absence of calcium ions. The physiological functions of the two routes of myosin adenosinetriphosphatase in muscle contraction. 12 89

The extent of actin polymerization has been studied for samples in which the bound nucleotide of the actin was ATP, ADP, or an analog of ATP that was not split (AMPPNP). The equilibrium constants for the addition of a monomer to a polymer end were determined from the concentration of monomer coexisting with the polymer. An analysis of these results concludes that the bound ATP on G-actin provides little energy to promote the polymerization of the actin. AMPPNP was incorporated into F-actin and the interaction of F-actin - AMPPNP with myosin was studied. F-actin - AMPPNP activated the ATPase of myosin to the same extent as did F-actin - ADP. However, the rate of superprecipitation was slower in the case of F-actin - AMPPNP than in the control.
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PMID:The bound nucleotide of actin. 12 84

Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5 mM, Vmax (minus trypsin) = 1.6 mumol-min-1-mg protein-1, Vmax (plus trypsin) = 2.44 mumol-min-1-mg protein-1; for the soluble ATPase: [S0.5] = 1.2 mM, Vmax (-trypsin) = 4 mumol-min-1-mg protein-1; Vmax (+ trypsin) = 6.6 mumol-min-1-mg protein-1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mM, respectively. Mg2+ saturation at constant ATP (8 mM) revealed michealian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 +/- 0.1 mM for the membrane-bound ATPase and 1.3 +/- 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 mM) inhibited the membrane-bound enzyme by 60-70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20 muM) inhibited both states of E. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.
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PMID:Membrane bound and soluble adenosine triphosphatase of Escherichia coli K 12. Kinetic properties of the basal and trypsin-stimulated activities. 12 30

The purpose of this investigation was twofold: (1) to identify and characterize the enzymatic ATP hydrolysis system of epithelial cilia, and (2) to develop a quantitative, biochemical test for the ciliotoxic cystic fibrosis (CF) factor based on inhibition of ATP utilization by ciliary preparations. Our rationale for selecting this system for CF factor analysis relates to the tight and essential mechanochemical coupling of functioning cilia. Using rabbit tracheal epithelium as the source, a high molecular weight (greater than 200,000) ATPase was identified, partially purified, and extensively characterized. The properties of this protein were similar to those observed in previous studies of others with flagellar and ciliary dynein (the motility-associated ATPase) isolated from microorganisms. Analysis of the pH profile revealed a broad range of high enzymatic activity between 6.5 and 9. Studies with potential cation activators showed that the enzyme is activated equally by either Ca2+ or Mg2+ in equimolar concentrations. No activation occurred in the presence of Zn2+, Na+, H+, or Na+ plus K+ and the effect of Mg2+ or Ca2+ was not inhibited by Na+, K+, or Na+ plus K+. The enzyme hydrolyzed Mg2+-containing solutions of UTP, CTP, and ADP at 51-54% the rate of ATP dephosphorylation, whereas Mg-deoxy-ATP was hydrolyzed 79% as effectively as ATP. Using a newly devised, analytical technique with [gamma-32P]ATP as the substrate, the ATP hydrolysis of various ciliary preparations from rabbit trachea and oyster gill (including motile suspensions) was monitored in the presence of sera from CF homo- and heterozygotes. Reproducible rates of ATP dephosphorylation averaging 27 nmol/min/mg protein were demonstrable with homogenates of ciliated epithelium. None of the test systems evaluated, however, were capable of demonstrating CF-related differences in ATPase activity or ATP utilization. Although these attemps have been unsuccessful thus far, the approach described in this report provides an example of an objective, quantitative, biochemical assessment of ciliary function.
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PMID:Determination and characterization of ciliary ATPase in the presence of serum from cystic fibrosis patients. 12 30

Ouabain-binding and phosphorylation of (Na+ mk+)-ATPase (EC 3.6.1.3) of the plasma membranes from kidney were investigated after treatment with N-ethylmaleimide or oligomycin. Either of these inhibitors brought about the following changes: the phosphoenzyme, formed in the presence of Na+, Mg2+ and ATP became essentially insensitive to splitting by K+ but was split by ADP. One mole of this ADP-sensitive phosphoenzyme bound one mole of ouabain but the enzyme-ouabain complex was less stable than in the native enzyme primarily because the rate of its dissociation increased. Ouabain was bound to the ADP-sensitive phosphoenzyme in the presence of Mg2+ alone and addition of inorganic phosphate enhanced both the rate of formation and the steady-state level of the enzyme-ouabain complex. The inhibitors did not affect the properties of this second type of complex. Both in the native enzyme and in the enzyme treated with the two inhibitors inorganic phosphate enhanced ouabain binding by phosphorylating the active center of the enzyme as shown (a) by mapping the labeled peptides from the enzyme after peptic digestion, (b) by inhibition of this phosphorylation with Na+ and (c) by the 1:1 stoichiometric relation between this phosphorylation and the amount of bound ouabain. Unlike the phosphoenzyme, the binding of ouabain remained sensitive to K+ in the enzyme treated with the inhibitors. K+ slowed ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding than to stimulate dephosphorylation. This finding is interpreted as being an indication of separate sites for K+ on the enzyme: a site(s) with high K+-affinity which stimulates dephosphorylation, another site(s) with moderate K+-affinity which inhibits ouabain-binding. Inhibitors may enhance formation of the ADP-sensitive phosphoenzyme by blocking interaction between K+ and the site(s) with high affinity.
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PMID:Ouabain-binding and phosphorylation of (Na+ + K+) ATPase treated with N-ethylmaleimide or oligomycin. 12 64

A preparation of ATPase with a high specific activity was isolated from the membrane of M. lysodeikticus. The enzyme was studied using UV-spectroscopy and circular dichroism. The homogeneity of the protein preparation was shown by gel electrophoresis. The catalytic properties of the enzyme were studied using steady state kinetic methods. The values of Km app. and kcat were determined to be 6-10(-4) and 6 mumoles/mg/min respectively. It is shown that ADP is an effective inhibitor of the ATPase reaction, and the inhibition activity increases in the presence of an excess of Ca2+. The nature of the rate dependence of the ATPase reaction on the concentration of the substrate and on Ca-ADP corresponds to a competitive type of inhibition with binding several molecules of Ca-ADP in the active site of the enzyme.
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PMID:[Adenosine triphosphatase from the membrane of Micrococcus lysodeikticus]. 12 72

The ATPase of matrix vesicles is not stimulated by calcium ions, nor do the vesicles have any capacity to metabolize glucose. ADPase of high activity is also present; thus vesicles cannot be a component of the conventional ATP cycle, in which energy is stored by phosphorylating ADP and released by hydrolyzing the resultant ATP. These results do not support speculations that matrix vesicles might function by concentrating calcium via an energy-dependent ion transport system such as those found in the plasma membrane and the sarcoplasmic reticulum. Matrix vesicles' alkaline phosphatase can be solubilized by treatment with certain detergents: sodium dodecyl sulfate (12 mM and 16 mM), cetylpyridinium chloride (14mM), and deoxycholic acid (DOC, 14 MM). The first two detergents denature the enzyme during storage whereas DOC does not. DOC will also solubilize ATPase and inorganic pyrophosphatase. Yields of the three enzymes are 85-95%. Dialysis of a DOC digest of vesicles removes DOC and 43% of protein, and also causes much of the alkaline phosphatase to become particulate once again.
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PMID:Matrix vesicles of bovine fetal cartilage: metabolic potential and solubilization with detergents. 12 41

Na+-K+-Dependent ATPase [EC 3.6.1.3] was preincubated with ATP in the presence of a high concentration of MgCl2, and the phosphorylated intermediate, EP, was formed by adding a high concentration of NaCl. The following results showed that EP was converted from an ADP-sensitive to an ADP-insensitive form by a single turnover of the ATPase reaction. 1. After initiating the reaction by adding NaCl, almost all the EP was at first sensitive to added ADP, but its sensitivity to ADP decreased with increase in the time interval between the additions of NaCl and of ADP. 2. Both in the presence and absence of KCl, the time course of the replacement of ADP-sensitive EP by ADP-insensitive EP coincided with the time course of the decomposition of EP after addition of EDTA.
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PMID:The pre-steady state of Na+-K+-dependent ATPase after addition of Na+ ions. Transition of the phosphorylated intermediate from an ADP-sensitive to an ADP-insensitive form. 12 69


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