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)

A method is described for purification of (Na+, K+)-ATPase which yielded approximately 60 mg of enzyme from 800 g of cardiac muscle with specific activities ranging from 340 to 400 mumol inorganic phosphate/mg protein per h (units/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a major 94 000 dalton polypeptide and four or five lesser components, one of which was a glycoprotein with an apparent molecular weight of 58 000. The enzyme preparation bound 600-700 pmol of [3H]ouabain/mg protein when incubated in the presence of either Mg2+ plus Pi, or Mg2+ plus ATP plus Na+, and incorporated more than 600 pmol 32P/mg protein when incubated with gamma-32P-labelled ATP in the presence of Mg2+ and Na+. The preparation is approximately 35% pure.
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PMID:Improved purification and partial characterization of (Na+, K+)-ATPase from cardiac muscle. 12 12

When ATP binds to myosin in the presence of Mg2+ there follows a rapid cleavage reaction to yield a myosin-product complex whose breakdown is rate-limiting in the overall adenosine triphosphatase reaction at 21 degrees and pH 8.0. Recent kinetic studies on this system have led to the proposal that the cleavage of ATP bound to myosin is reversible. This conclusion is based in part on the observation that when ATP is mixed with an excess of myosin active sites a small amount of tightly bound ATP exists whose life-time coincides with that of the myosin-product complex and implies these two species are in equilibrium during their decay. Previous oxygen exchange studies have shown that phosphate released as free product contains more than one oxygen atom from water. A rapid equilibration between myosin-bound ATP and a myosin-products complex can account for the extra water oxygen incorporation of the product phosphate. Such a model requires that the gamma-phosphoryl group of the bound ATP also exchanges its oxygen atoms with water. Results presented in this paper show that protein-bound ATP labeled in the three terminal oxygen atoms of the gamma-phosphoryl group with 18O exchanges about 75% of its label within 2 s of binding to the active site of myosin. This result provides chemical evidence for a model in which bound ATP undergoes a reversible reaction with water. Incomplete exchange may arise from kinetic and/or structural restraints on the mechanism and plausible models are discussed.
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PMID:Oxygen exchange in the gamma-phosphoryl group of protein-bound ATP during Mg2+-dependent adenosine triphosphatase activity of myosin. 12 49

Isoproterenol (ISO) in a single dose of 7.5 mg/kg b.w. i.v was found to produce alterations of cardiac metabolism in dogs. After 2 h, high energy phosphate stores and glycogen were reduced, whereas the levels of lactate, pyruvate, the lactate/pyruvate ratio and myofibrillar ATPase activity were elevated. Ca2+ -accumulation by sarcoplasmic reticulum (SR) and mitochondria were increased (P less than 0.01). After 24 h, a partial recovery in the parameters followed could be observed. Only myofibrillar ATPase activity and the Ca2+ -uptake by SR and mitochondria were lowered. When K,Mg-ASP was administered, i.v. concurrently with ISO, myofibrillar ATPase and Ca2+ -accumulation by SR did not differ from controls 2 h after ISO application. Also the other parameters exhibited a tendency to improve (P less than 0.01), but did not reach control levels. 24 h after ISO application we could observe a similar effect of K,Mg-ASP in the prevention of Ca2+ -overload accompanying metabolic changes.
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PMID:K+, MG2+-aspartate (KMg-ASP)--mediated prevention of isoproterenol(ISO)-induced metabolic changes in the myocardium. 12 87

(Na+ + K+)-activated ATPase in beef brain microsomes is inactivated by the disulfide of thionosine tri[gamma-32P]phosphate, an ATP analog. The inactivation of the enzyme, which is accompanied by an incorporation of radioactivity into the membrane protein, is abolished by ATP or dithiothreitol. Since dithiothreitol restores the activity of (Na+ + K+)-ATPase, which had previously been inactivated by this ATP analog, it is concluded that thionosine triphosphate disulfide reacts with a sulfhydryl group in the ATP binding site of (Na+ + K+)-activated ATPase.
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PMID:Disulfide of thionosine triphosphate, an ATP-analog inactivating (Na+ + K+)-ATPase. 12 99

In order to study the "sidedness" of the ligands of the Na+, K+-ATPase in the phosphorylation from [32P]ATP, tight vesicles were prepared from guinea pig kidney and partially purified by a two-stage sucrose and Ficoll gradient centrifugation procedure. These vesicles were derived presumably from plasma membrane fragments resealed after the initial disruption of the cells during homogenization. Tightness of the vesicles was estimated according to activation by the nonionic detergent, Triton X-100. Treatment with Triton X-100 increased both the activity of the Na+, K+-ATPase and its Na+-dependent phosphorylation from [32P]ATP at least three-fold. Activation of both functions also appeared when the vesicles were shocked osmotically. These results suggest that the preparation contains a major population of tight normal vesicles (approximately 75%) in which the phosphorylation site faces the intravesicular solution. In the response to ouabain breakdown of the phosphoenzyme was inhibited in vesicles treated with Triton X-100 but not in intact ones as if ouabain could not get to its binding site. Correspondingly in phosphorylation from ATP pretreatment with ouabain in the presence of inorganic phosphate produced less inhibition in intact vesicles than in those disrupted with Triton X-100 beforehand. These data suggest the presence of an everted vesicle fraction in the preparation (approximately 20%). Apparently only a small fraction of the vesicles was leaky. In the everted vesicles the action of K+ on the phosphoenzyme was slow. In order to accelerate the dephosphorylation in intact vesicles as effectively as in disrupted ones, K+ had to be added before the start of phosphorylation. This supports the view that K+ was acting from the side of the membrane opposite to that where the gamma-phosphoryl group was accepted from ATP.
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PMID:Tightness and orientation of vesicles from guinea-pig kidney estimated from reactions of adenosine triphosphatase dependent on sodium and potassium ions. 12 64

In rat liver, homogenized and fractionated, ATPase activity was studied both histochemically and biochemically in the presence of lead ions and with addition of cysteine, EDTA, 2,4-DNP, sodium cyanide, sodium fluoride and p-chloromercuribenzoate. None of the inhibitors concerned permitted to obtain a complete inhibition of ATP hydrolysis. A practically complete inhibition of ATPase was possible only through heat inactivation or after double prefixation with formaldehyde and ethanol. Nonenzymatic ATP hydrolysis plays a negligible role in ATPase histochemistry since phosphate yield taking place due to lead action is insignificant against enzymatic hydrolysis of ATP.
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PMID:[Inhibition and stimulation of ATPase activity and the nonenzymatic hydrolysis of ATP in electron histochemistry (author's transl)]. 12 80

Effect of inorganic phosphate (4 X 10(-3) M) on Ca++-accumulation was examined in kidney cortex mitochondria. Ca++-accumulation of rat kidney cortex mitochondria was slightly influenced by inorganic phosphate. On the other hand, dog kidney cortex mitochondria did not accumulate calcium from the incubation medium until the inorganic phosphate had been added. Ca++-accumulation of rabbit kidney cortex mitochondria was markedly stimulated by inorganic phosphate. When ethacrynic acid was added to the reaction medium in the absence of inorganic phosphate, Ca++-accumulation of rat kidney cortex mitochondria was depressed and the decrease in calcium content of rabbit and dog kidney cortex mitochondria was enhanced. In the presence of inorganic phosphate, the inhibition of Ca++-accumulation by ethacrynic acid was observed only on dog kidney cortex mitochondria. Subsequently, the effect of inorganic phosphate (4 X 10(-4) M) and ethacrynic acid (1 X 10(-4) M on Ca++-ATPase was examined in kidney cortex mitochondria. The low concentration of inorganic phosphate (4 X 10(-4) M) activated Ca++-ATPase of kidney cortex mitochondria in all animal species. The greatest activation of Ca++-ATPase occurred in rabbits, but the activity of the enzyme was lower than that in rats and dogs. Inhibition of Ca++-ATPase by ethacrynic acid was depressed by the addition of inorganic phosphate in kidney cortex mitochondria of experimental animals. Ca++-accumulation may be regulated through the stimulating effect of inorganic phosphate and the inhibitory effect of ethacrynic acid on Ca++-ATPase in kidney cortex mitochondria. Species difference in ethacrynic acid effect on Ca++-accumulation in kidney cortex mitochondria of rats, rabbits and dogs is discussed.
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PMID:Effect of diuretics on ion transport of kidney cortex mitochondria. III. Species difference in calcium accumulation and in ethacrynic acid effect. 12 57

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, KD = 1-2 muM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (KI = 240-300 muM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This "tightly bound" ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetics studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, Pi can maintain the active form of the enzyme.
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PMID:Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate. 12 85

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

A particulate subcellular fraction from Escherichia coli K-12 induced in anaerobic sn-glycerol 3-phosphate (G3P) dehydrogenase and fumarate reductase can catalyze under anaerobic conditions the transfer of hydrogens from G3P to fumarate, with attendant generation of high-energy phosphate. The phsophorylation process is more sensitive than the transhydrogenation process to inhibition by the detergent Triton X-100. The same is true with respect to sensitivity to sodium azide, carbonyl cyanide m-chlorophenylhydrazone and N,N'-dicyclohexylcarbodiimide. Such a preparation derived from cells with beta-galactoside permease can accumulate thiomethyl beta-D-galactoside anaerobically, and the accumulation can be stimulated twofold by adding G3P and fumarate. Mutants lacking the membrane-associated Mg2+-dependent adenosine triphosphatase cannot grow anaerobically on glycerol with fumarate as the hydrogen acceptor, although they can grow aerobically on glycerol alone.
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PMID:Anaerobic energy-yielding reaction associated with transhydrogenation from glycerol 3-phosphate to fumarate by an Escherichia coli system. 12 85


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