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)

The pH-activity curve of heavy meromyosin ATPase [EC 3.6.1.3] was measured at various temperatures. The pH-activity curve at higher temperatures showed a maximum at low pH and a minimum at pH 7 to 8 as has been already reported. At lower temperatures it was sigmoidal in shape, similar to a simple dissociation curve of pKa 6 to 7. The pH-activity curve at intermediate temperatures appeared to be inbetween the two extreme shapes. These changes in pH-activity curve with temperature were found to be common in the presence of divalent cations such as Mg2+, Mn2+, and Ca2+. The ATPase mechanism may be identical in the presence of any divalent cation, and the rate determining step revealing the steady state rate alters by changing the temperature. The transition temperatures estimated at pH 8 were 10 degrees, 8 degrees, and about 5 degrees in the presence of MnCl2, CaCl2, and MgCl2, respectively. The difference in the temperature coefficients above and below the transition temperature was most distinct in the presence of MnCl2, and vague in the presence of CaCl2. A similar change of pH-activity curve with temperature was found with heavy meromyosin ITPase in the presence of MgCl2.
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PMID:Temperature induced transition of the pH-activity curve of heavy meromyosin adenosine triphosphatase and inosine triphosphatase. 1 40

The rate of enzymic reaction of ATP, ITP, GTP with myosin is studied in the presence of potassiu, ammonium and calcium ions in H2O--D2O solutions. There is no kinetic isotope effect of ITPase and GTPase reaction in the neutral pH region (VHVD = 1). The value VH/VD for the ATPase reaction in the pH range from 6.5 to 8.5 with all cations studied varies from 1.05 to 1.26. Such changes of myosin enzymic activity in D2O infer that small changes in the interaction of subunits is not the decisive one in the regulation of myosin ATPase. The equality of isotope effects in potassium salts and ammonium solution suggests that a specific effect of ammonium ion as a proton donor affects the ATPase reaction of myosin. The relationship between the value of isotope effect and D2O concentration in solution in non-linear. The shape of concentration curve suggests essential conformational changes of myosin during ATP hydrolysis.
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PMID:[Enzyme activity of myosin activated by different cations in a mixed H2O--D2O solvent]. 3 22

Investigations have been made of the kinetic effects of the antibiotic aurovertin on the ATPase and ITPase activity of isolated rat liver mitochondrial ATPase. Unusual patterns of inhibition, decreasing slope, and increasing y-intercept values of double reciprocal plots, were observed with Mg-ATP as the substrate under various conditions. Under specified conditions, aurovertin stimulated hydrolysis of MgATP. The inhibition of MgITP hydrolysis was uncompetitive. Aurovertin eliminated the HCO3-minus stimulation of MgATP hydrolysis. The implications of these findings for the mechanism of mitochondrial ATPase are briefly discussed.
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PMID:Influence of aurovertin on mitochondrial ATPase activity. 12 77

A partially purified soluble ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pea cotyledon mitochondria was characterized. Inhibition patterns with azide, NaF, and cold, and a stimulation by 2,4-dinitrophenol were typical of F1-ATPases from mammalian mitochondria. The enzyme hydrolysed GTP, ITP, and ATP, but not CTP, UTP, ADP, or IDP. ATPase and ITPase activities were strongly inhibited by ADP and to a lesser extent by IDP. Distinctive properties of the pea mitochondrial enzyme were activation by high concentrations of CaCl2 and stimulation by NaCl.
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PMID:Partial characterization of a soluble ATPase from pea cotyledon mitochondria. 14 76

Membrane phosphorylation and nucleoside triphosphatase activity of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle were studied using ATP and ITP as substrates. The Ca2+ concentration was varied over a range large enough to saturate either the high affinity Ca2+-binding site or both high and low affinity binding sites. In intact vesicles, which are able to accumulate Ca2+, the steady state level of enzyme phosphorylated by either ATP or ITP is already high in 0.02 mM Ca2+ and does not vary as the Ca2+ concentration is increased to 10 mM. Essentially the same pattern of membrane phosphorylation by ATP is observed when leaky vesicles, which are unable to accumulate Ca2+, are used. However, for leaky vesicles, when ITP is used as substrate, the phosphoenzyme level increases 3- to 4-fold when the Ca2+ concentration is raised from 0.02 to 20 mM. When Mg2+ is omitted from the assay medum, the degree of membrane phosphorylation by ATP varies with Ca2+ in the same way as when ITP is used in the presence of Mg2+. Membrane phosphorylation of leaky vesicles by either ATP or ITP is observed in the absence of added Mg2+. When these vesicles are incubated in media containing ITP and 0.1 mM Ca2+, addition of Mg2+ up to 10 mM simultaneously decreases the steady state level of phosphoenzyme and increases the rate of ITP hydrolysis. When ATP is used, the addition of 10 mM Mg2+ increases both the steady state level of phosphoenzyme and the rate of ATP hydrolysis. When the Ca2+ concentration is raised to 10 or 20 mM, the degree of membrane phosphorylation by either ATP or ITP is maximal even in the absence of added Mg2+ and does not vary with the addition of 10 mM Mg2+. In these conditions the ATPase and ITPase activities are activated by Mg2+, although not to the level observed in 0.1 mM Ca2+. An excess of Mg2+ inhibits both the rate of hydrolysis and membrane phosphorylation by either ATP or ITP.
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PMID:Calcium and magnesium regulation of phosphorylation by ATP and ITP in sarcoplasmic reticulum vesicles. 18 11

The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p'-fluorosulfonylbenzoyl-8-azidoadenosine (8-N3-FSBA) with an apparent Kd of 0.47 mM at pH 8.0 and 23 degrees C in the absence of light. Irradiation of dark-inactivated enzyme with long-wavelength UV light produced cross-linked dimers and, to a lesser extent, trimers made up of alpha and beta subunits. Two major radioactive peptides were resolved by high-performance liquid chromatography from tryptic digests of MF1 which had been inactivated with 8-N3-FSB[3H]A at pH 8.0 in the dark. Sequence analysis revealed that one contained Tyr-beta 368 and the other contained His-beta 427 which were labeled in the ratio of 18:15. Sequence analysis of radioactive tryptic peptides isolated from digests of irradiated MF1 derivatized with 8-N3-FSB[3H]A showed that photolysis induced cross-linking of His-427 to Tyr-345 within the same beta subunit in high yield. When MF1 derivatized with 8-N3-FSB[3H]A was irradiated in the presence of beta-mercaptoethanol, alpha-beta cross-links were eliminated, whereas those between His-beta 427 and Tyr-beta 345 were unaffected. Analysis of radioactive peptides in tryptic digests of MF1 derivatized with 8-N3-FSB[3H]A and then irradiated in the presence or absence of beta-mercaptoethanol showed that the nitrene generated from reagent attached to Tyr-beta 368 participates in formation of alpha-beta cross-links in the absence of beta-mercaptoethanol. Therefore, the nitrene generated from reagent tethered to His-beta 427 is shielded from solvent and reacts with the side chain of Tyr-beta 345. In contrast, the nitrene generated from reagent attached to Tyr-beta 368 is exposed to solvent, but in the absence of scavengers reacts with side chains present in the alpha subunit. Irradiation of MF1, partially inactivated with 8-N3-FSBA, led to loss of residual ATPase activity without affecting residual ITPase activity. The amount of photoinactivation was greater when partial dark inactivation was performed at pH 6.9, where modification of His-beta 427 predominates, than when performed at pH 8.0, where modification of Tyr-beta 368 predominates. This suggests that cross-linking of His-beta 427 to Tyr-beta 345, and not cross-linking of alpha and beta subunits, is responsible for the augmented inactivation induced by irradiation.
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PMID:Irradiation of the bovine mitochondrial F1-ATPase previously inactivated with 5'-p-fluorosulfonylbenzoyl-8-azido-[3H]adenosine cross-links His-beta 427 to Tyr-beta 345 within the same beta subunit. 132 8

The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p-fluorosulfonylbenzoylethenoadenosine (FSB epsilon A) with pseudo-first order kinetics. The dependence of the rate of inactivation on the concentration of FSB epsilon A revealed an apparent Kd of 0.25 mM. ATP and ADP, and to a lesser extent, ITP and IDP provide partial protection against inactivation by the reagent. Isolation and sequence analysis of major radioactive fragments in peptic or cyanogen bromide digests of MF1 inactivated with [3H]FSB epsilon A indicate that modification of Tyr-alpha 244 is associated with the loss of activity observed. Assessment of the amount of Tyr-alpha 244 derivatized with [3H]FSB epsilon A at specific points during inactivation of the ATPase indicates that maximal inactivation is achieved on modification of this residue in slightly greater than one copy of the alpha subunit. The following characteristics of inactivation of MF1 by FSB epsilon A have also been determined. (a) The rate of inactivation of ITPase activity by FSB epsilon A is 1.4 times greater than that observed for inactivation of ATPase activity under identical conditions. (b) After maximally inactivating the capacity of MF1 to hydrolyze saturating ATP with FSB epsilon A, the modified enzyme retained its capacity to hydrolyze substoichiometric ATP. (c) Inactivation of the ATPase by FSB epsilon A is accelerated by Pi. In each of the above characteristics, MF1 modified by FSB epsilon A resembles enzyme inactivated with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) more than it does enzyme inactivated with 5'-p-fluorosulfonylbenzoylinosine (FSBI). Furthermore, prior inactivation of MF1 with FSBA completely prevents labeling of Tyr-alpha 244 with [3H]FSB epsilon A, whereas prior inactivation of the enzyme with FSBI does not. Since a single catalytic site is modified when FSBI inactivates MF1 whereas three noncatalytic sites are modified when it is maximally inactivated with FSBA, it is concluded that FSB epsilon A also modifies noncatalytic sites.
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PMID:Tyrosine alpha 244 is derivatized when the bovine heart mitochondrial F1-ATPase is inactivated with 5'-p-fluorosulfonylbenzoylethenoadenosine. 213 76

Phenotypic revertants have been selected from mutants of the yeast Schizosaccharomyces pombe devoid of either alpha or beta subunits of mitochondrial ATPase-ATPsynthase. In contrast to parental mutants, phenotypic revertants are able to grow on glycerol respiratory medium and show immunodetectable alpha and beta subunits. However, growth and cellular respiration are only partially restored as compared to the wild strain, indicating that the recovered subunits are mutated. ATPase activity of revertant submitochondrial particles shows markedly different parameters: more acidic optimal pH, absence of bicarbonate activation and decreased sensitivity to azide inhibition in the alpha subunit-modified R3.51. Opposite differences are observed in the beta subunit-modified R4.3: more alkaline optimal pH, much higher bicarbonate activation, and increased sensitivity to azide. The ITPase activity of R4.3 submitochondrial particles is also more sensitive to azide as compared to the wild strain. ATPase activity of purified F1 also exhibits marked differences: loss of bicarbonate-sensitive negative cooperativity, decreased sensitivity to both ADP and azide inhibitions in the R3.51 revertant. On the contrary, increased negative cooperativity and increased sensitivity to both ADP and azide inhibitions are observed for the R4.3 revertant enzyme which in addition exhibits a much lower maximal rate. The beta subunit-mutation of R4.3 also increases the sensitivity of ITPase activity to tripolyphosphate inhibition, whereas the alpha subunit-mutation of R3.51 is without any effect. Soluble F1 with beta subunit-mutation is very sensitive to high ammonium sulfate concentrations required for enzyme precipitation and concentration and known to partially deplete the enzyme from its endogenous nucleotides. On the contrary, poly(ethylene)glycol is very efficient for preparing from any strain a pure and very stable enzyme retain-ing high amounts of endogenous nucleotides. The R4.3 revertant F1 retains even more nucleotides than the wild-strain F1 and is much less sensitive to high iodide concentrations which favor enzyme dissociation and precipitation. The tryptophan intrinsic fluorescence of F1 is modified by both mutations that increase the maximal emission intensity. The most important effect is produced by beta subunit-mutation which decreases the quenchable fraction, one-third to one-half tryptophans being no longer accessible to iodide. The overall results suggest that both mutations modify enzyme-nucleotide interactions: the alpha subunit-mutation of R3.51 would favor ADP release by lowering interactions with the adenine moiety, whereas the beta subunit-mutation of R4.3 would lower ADP release by strengthening interactions with the phosphate chain moiety.
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PMID:Structure-function relationships of mitochondrial ATPase-ATPsynthase using Schizosaccharomyces pombe yeast mutants with altered F1 subunits. 252 9

This study examined the nature of the metal-nucleotide complexes which serve as substrates, products, and intermediates in the beef heart mitochondrial ATPase reaction. The two methods employed involved the use of phosphorothioate ATP analogs as substrates in the presence of Mg2+ or Cd2+ and the use of substitution inert Cr X ATP complexes (the isolated diastereomers of the bidentate complexes) along with the newly synthesized Cr X ITP complexes as inhibitors of both the F1-ATPase and F1-ITPase activities. Little stereoselectivity was observed in the inhibition of F1-ATPase and F1-ITPase activities by the isolated diastereomers of beta,gamma-bidentate CrATP, while the inhibition by the delta,alpha,beta-bidentate CrADP diastereomer was greater than that of the lambda epimer. gamma-Monodentate CrITP was a weak inhibitor of both the ATPase and ITPase activities, whereas beta,gamma-bidentate CrITP failed to show any inhibition at all up to a concentration of 3.2 mM. When adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) was used as the substrate, (VmSp]/(Vm(Rp] with Mg2+ present was 2.7 at 31 degrees C and 3.5 at 13 degrees C. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Mg2+ present were 15.3 at 31 degrees C and 73.3 at 13 degrees C. With Cd2+ present, the (Vm(Sp]/(Vm(Rp] ratios were 0.81 and 0.65 at 31 and 13 degrees C, respectively. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Cd2+ present were 1.17 at 31 degrees C and 1.34 at 13 degrees C. The large activation energy observed for the isomers of CdATP beta S was not observed for MgATP beta S, MgATP, or CdATP. The Vm for Cd adenosine 5'-O-thiotriphosphate (ATP gamma S) hydrolysis was the largest of all the metal-phosphorothioate nucleotide complexes, while that for MgATP gamma S was the smallest. The results are interpreted in terms of a catalytic model for F1-catalyzed nucleotide hydrolysis describing metal-nucleotide chelation during the reaction.
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PMID:Metal-nucleotide structural characteristics during catalysis by beef heart mitochondrial F1. 286 Jan 7

Characteristics of Ca2+ uptake were studied in a vesicular preparation of proximal tubule plasma membranes from rabbit kidney and compared with the properties of both membrane-bound and solubilized Ca2+-ATPase activities. Calcium uptake required both ATP and MgCl2 and revealed two kinetic components with respect to Ca2+ concentration requirements, one with a high affinity for Ca2+ (1.8 microM), operative in the range of cytosolic Ca2+ activity, and one with a low affinity for Ca2+ (250 microM) which may become active only at abnormally high cytosolic Ca2+ concentrations. The high- and low-affinity components were stimulated to similar extents by phosphate, and required similar concentrations of ATP (0.6 mM) for half-maximal activity. The amount of membrane-bound phosphoenzyme formed from ATP in the presence of Ca2+ was the same regardless of whether only one or both sites were saturated, suggesting that occupancy of the second Ca2+ binding site accelerates the enzyme turnover. Inhibition of Ca2+ transport by Na+ was reversed by the addition of ouabain or an ATP-regenerating system, indicating that this inhibitory effect of Na+ on Ca2+ uptake may be due to the accumulation of ADP in the medium as a result of Na+ pump activity. Low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and valinomycin (2.5 and 1 microM, respectively) were without effect on Ca2+ uptake in the presence of phosphate, whereas higher concentrations of the ionophores (200 and 100 microM, respectively) reduced uptake by 60% or more. The calmodulin antagonist 48/80 also reduced Ca2+ uptake with half-maximal effectiveness at 100 micrograms/ml. None of these drugs affected either ATPase activity or the EGTA-induced Ca2+ efflux from preloaded vesicles. The Ca2+ dependence of ATP hydrolysis by the membrane-bound enzyme preparation was similar to that observed for Ca2+ uptake by the vesicles. However, with solubilized enzyme, concentrations of Ca2+ similar to that found in the plasma reduced Ca2+-stimulated ATP hydrolysis to one-half of its maximal rate. This indicates that peritubular Ca2+ may play a role in the regulation of Ca2+ transport across the tubular epithelium. ATP could not be replaced by ITP as a substrate for Ca2+ uptake, and the (Ca2+ + Mg2+)ITPase activity of soluble enzyme was 25-fold lower than in the presence of ATP. This is an indication that the active Ca2+ pumping mechanism in proximal tubules is critically dependent on the nucleoside moiety of the substrate.
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PMID:Comparison between calcium transport and adenosine triphosphatase activity in membrane vesicles derived from rabbit kidney proximal tubules. 300 27


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