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Query: UNIPROT:P20020 (adenosine triphosphatase)
 3,299 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of ouabain, a specific sodium-potassium dependent adenosine triphosphatase (Na+-K+-ATPase) inhibitor, on antigen-induced histamine release was studied using guinea pig lung fragments sensitized in vitro with rabbit antibodies against bovine serum albumin. Histamine was assayed spectrofluorometrically. When sensitized tissue had been preincubated with ouabain (less than or equal to 1.0 x 10(-4) M) for 10 min prior to antigenic challenge, release of histamine was significantly inhibited (maximum 54%, p less than 0.001, N=9, paired t test). The most significant inhibition was obtained near the optimal concentration of antigen. The inhibition was dependent on the length of preincubation (less than or equal to 20 min), and was partially reversible upon washing the tissue removing the ouabain. Ouabain did not seem to prolong the duration of the histamine release process. Increase in potassium ion (less than or equal to 1.1 x 10(-2)M) inhibited the histamine release and had additive effects to ouabain action. Dibutyryl cyclic AMP (less than or equal to 5 x 10(-3) M), which could enhance the release, strongly antagonized the inhibition. Glucose removal from the medium did not abolish the ouabain effect. The results seem to indicate that immunologic release of histamine is under the influence of the membrane Na+-K+-ATPase activity.
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PMID:Inhibition of antigen-induced histamine release by ouabain. 5 30

Radioactive adenosine triphosphate was synthesized transiently from adenosine diphosphate and radioactive inorganic phosphate by sodium and potassium adenosine triphosphatase from guinea pig kidney. In a first step, K+-sensitive phosphoenzyme was formed from radioactive inorganic phosphate in the presence of magnesium ion and 16 mM sodium ion. In a second step the addition to the phosphoenzyme of adenosine diphosphate with a higher concentration of sodium ion produced adenosine triphosphate. Recovery of adenosine triphosphate from the phosphoenzyme was 10 to 100% in the presence of 96 to 1200 mM sodium ion, respectively. Potassium ion (16mM) inhibited synthesis if added before or simultaneously with the high concentration of sodium ion but had no effect afterward. The half-maximal concentration for adenosine diphosphate was about 12 muM. Ouabain inhibited synthesis. The ionophore gramicidin had no significant effect on the level of phosphoenzyme nor on the rate nor on the extent of synthesis of adenosine triphosphate. The detergent Lubrol WX reduced the rate of phosphoenzyme break-down and the rate of synthesis but did not affect the final recovery. Phospholipase A treatment inhibited synthesis. In a steady state, the enzyme catalzyed a slow ouabain-sensitive incorporation or inorganic phosphate into adenosine triphosphate. These results and other suggest that binding of sodium ion to a low affinity site on phosphoenzyme formed from inorganic phosphate is sufficient to induce a conformational change in the active center which permits transfer of the phosphate group to adenosine diphosphate.
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PMID:Synthesis of adenosine triphosphate and exchange between inorganic phosphate and adenosine triphosphate in sodium and potassium ion transport adenosine triphosphatase. 12 28

Ouabain interaction with a possible pharmacologic receptor, Na+, K+-adenosine triphosphatase (Na+, K+-ATPase), has been assessed by continual perfusion of canine hearts with various concentrations of both unlabeled and 3-H-ouabain. A positive dose-related correlation between enzyme inhibition, increased contractile force and drug binding to the enzyme has been established. The complex formed between 3-H-oubain and Na+, K+-ATPase in vivo appears to have the same characteristics as that formed in vitro, suggesting that the nature of both complexes is the same. These data are consistent with the concept that Na+, K+-ATPase may be an important pharmacologic receptor for cardiac glycosides.
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PMID:The nature of the transport adenosine triphosphatase-digitalis complex. VIII. The relationship between in vivo-formed (3-H-ouabain-Na+, K+-adenosine triphosphatase) complex and ouabain-induced positive inotropism. 12 84

The chemical properties of two highly purified preparations of (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) and their subunits have been compared. One preparation is derived from the rectal gland of the spiny dogfish shark, Squalus acanthias and the other preparation is derived from the electric organ of the electric eel, Electrophorus electricus. Ouabain binding and phosphorylation from [gamma-32-P]ATP for both enzymes ranged from 4000 to 4300 pmol per mg of protein. This gives a stoichiometry for ouabain binding and phosphorylation of 1:1 for both enzymes. The molar ratios of catalytic subunit to glycoprotein was 2:1 for both enzymes, suggesting a minimum molecular weight of 250, 000, which agrees with the molecular weight obtained by radiation inactivation. Assuming that only one of the two catalytic subunits is phosphorylated and binds ouabain per (sodium + potassium)-activated adenosine triphosphatase molecule the data on phosphorylation and ouabain binding also give a molecular weight of 250, 000. The data on phosphorylatiion, ouabain binding, subunit composition, and molecular weight based on radiaion inactivation are thus all internally consistent. A technique has been developed for isolation of pure catalytic subunit and glycoprotein in good yields by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A variety of chemical studies have been carried out with the purified subunits. The amino acid composition of the catalytic subunit was different from that of the glycoprotein, but the amino acid composition of each of the two subunits was essentially the same for both species. However, the NH2-terminal amino acid for the catalytic subunit was alanine for the rectal gland enzyme and serine for the electric organ enzyme, suggesting some differencesin amino acid sequences for the two species. The NH2-terminal amino acid for the glycoprotein was alanine for the two species. The glycoproteins from both species contained the same carbohydrates but in quite differing amounts. The carbohydrates were glucosamine, sialic acid, fucose, galactose, mannose, and glucose. The release of all the sialic acid from the electric organ enzyme and the release of 40% of the sialic acid from the rectal gland enzyme did not affect (sodium + potassium)-activated adenosine triphosphatase activity. Both enzymes contained the following phospholipids, which accounted for 98 to 100% of the total phospholipid phosphorus: sphingomyelin, lecithin, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylserine, the amount of any phospholipid per mg of enzyme as well as the total phospholipid content were quite different for the two enzymes.
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PMID:Molecular properties of purified (sodium + potassium)-activated adenosine triphosphatases and their subunits from the rectal gland of Squalus acanthias and the electric organ of Electrophorus electricus. 12 22

The purpose of this work was to test the previously suggested hypothesis that the inhibitory effect of ouabain on lactate production in human red cells is due to an interaction between phosphoglycerate kinase and (Na+ + k+)-activated adenosine triphosphatase (Na+,K+ATPase). An antibody to red cell phosphoglycerate kingase caused complete inhibition of the purified enzyme, whereas a portion of the phophoglycerate kinase activity of the red cell membranes was resistant to the antibody. When increasing amounts of the purified enzyme were added to the membranes, the antibody-resistant portion of the activity increased. The effects of the antibody and ouabain on lactate production from fructose-6,6-diphosphate in red cell hemolysates were studied. Ouabain, at a maximally effective concentration, produced about 30% inhibition of lactate formation. This value was doubled in the presence of the antibody. Red cell membranes, and rat brain Na+,K+-ATPase, did not catalyze the hydrolysis of 1,3-diphosphoglycerate. Ouabain did not affect the reactions of the Rapport-Luebering pathway of the red cells. These findings provide further support for the view that in red cells a membrane pool of phosphoglycerate kinase is oriented in the vicinity of Na+,K+-ATPase in a way that the product of each enzyme may be used as the immediate substrate of the other and that ouabain inhibits glycolysis by removing the regulatory effect of Na+,K+-ATPase on that portion of glycolysis which is channeled through this pool of phosphoglycerate kinase.
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PMID:Studies on the mechanism of inhibition of the red cell metabolism by cardiac glycosides. 12 26

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

A plasma membrane fraction of HeLa S3 cells, consisting of ghosts, is characterized more fully. A simple procedure is described which permits light and electron microscope study of the plasma membrane fraction through the entire depth of the final product pellet and through large areas parallel to the surface. Contamination by nuclei is 0.14%, too little for DNA detection by the diphenylamine reaction. Contamination by rough endoplasmic reticulum and ribosomes is small, a single ghost containing about 3% of the RNA in a single cell. Mitochondria were not encountered. Electron microscopy also shows (a) small vesicles associated with the outer surface of the ghosts, and (b) a filamentous web at the inner face of the ghost membrane. Sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis shows that of the many Coomassie Blue-stained bands two were prominent. One, 43,000 daltons, co-migrated with purified rabbit muscle actin and constituted about 7.5% of the plasma membrane protein. The other major band, 34,000 daltons, was concentrated in the plasma membrane fraction. Two major glycoproteins detected by autoradiography of [14C]fucose-labeled glycoproteins on the gels, had apparent molecular weights of 35,000 daltons and 32,000 daltons. These major bands did not stain with Coomassie Blue. There were many other minor glycoprotein bands in the 200,000- to 80,000-dalton range. Ouabain-sensitive, Na+, K+-adenosine triphosphatase (ATPase) activity of the ghost fraction is purified 9.1 (+/- 2.2) times over the homogenate; recover of the activity is 12.0 (+/- 3.8%) of the homogenate. Enrichment and recovery of fucosylglycoprotein parallel those for ouabain-sensitive Na+, K+-ATPase activity. Fucosyl glycoprotein is recovered more than the enzyme activity in a smooth membrane vesicle fraction probably containing the bulk of plasma membrane not recovered as ghosts.
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PMID:Further characterization of HeLa S3 plasma membrane ghosts. 14 66

1 Intracellular potentials were recorded in driven left atria from reserpine-treated rabbits. Guanethidine 2 X 10(-5) M slightly increased Vmax and shortened the total duration (TD) of the action potential (AP) without causing hyperpolarization. For the first 30 min after 4 X 10(-4) M, Vmax increased without hyperpolarization and AP height increased slightly. Thereafter, Vmax and height decreased with a slight and gradual depolarization. This depolarization was irreversible. TD was increased after 15 minutes. Guanethidine 2 X 10(-3) M initially decreased Vmax and height before causing depolarization. 2. Pretreatment with tetrodotoxin (TTX) 1.6 X 10(-7) M prevented or reversed the initial increases in Vmax, height and TD induced by guanethidine (4 X 10(-4) M). 3 TTX 3.1 to 6.2 X 10(-6) M, added 15 or 30 min after guanethidine 4 X 10(-4) M, delayed or prevented depolarization by guanethidine. 4 Ouabain 10(-5) M incubated for 20 and 90 min greatly inhibited Na+, K+-adenosine triphosphatase and K+-phosphatase activities; guanethidine was without effect. 5 Guanethidine probably increases resting sodium permeability after the promotion of increases in sodium permeability during the AP. High doses of the drug decrease sodium permeability during the AP.
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PMID:Action of guanethidine on rabbit atrial membranes. 20 4

Effects of vanadate on ouabain binding and inhibition of sodium and potassium adenosine triphosphatase (Na+ + K+)-ATPase) were investigated under various ionic conditions. 1. Vanadate facilitated ouabain binding to (Na+ + K+)-ATPase in the presence of Mg2+ and this facilitation was partially reversed by catechol. 2. Vanadate antagonized the ability of high concentrations of NaCl to inhibit ouabain binding in the presence of magnesium. 3. Ouabain binding to the vanadate-enzyme complex, formed from magnesium and vanadate, was more sensitive to depression by potassium than that to the phosphoenzyme formed from magnesium and inorganic phosphate. 4. Preincubation of (Na+ + K+)-ATPase with vanadate in the presence of magnesium initially formed a potassium-insensitive complex as shown by a rapid initial rate of ouabain binding. However, within 5 min potassium overcame the vanadate potentiation of ouabain binding regardless of the order in which it was added to the reaction mixture. 5. Under conditions of enzyme turnover, vanadate failed to antagonize the inhibitory power of ouabain despite the presence of a high concentration of potassium. This suggests a possible relationship between the sensitivity of the sodium pump in various tissues to the cardiac glycosides and intracellular vanadate concentrations.
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PMID:Effects of vanadate on ouabain binding and inhibition of (Na+ + K+)-ATPase. 22 60

1. Explants of mammary tissue from pseudopregnant rabbits were cultured at 37 degrees C in air for 24-48h in Medium 199 buffered with 20mm-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid]. The medium contained insulin and corticosterone, or insulin, corticosterone and sheep prolactin in the presence or absence of ouabain, an inhibitor of Na(+)/K(+)-dependent adenosine triphosphatase. The responses of explants were assessed histologically, by measuring the tissue concentration of K(+), and by rates of synthesis of RNA, protein and fatty acids. The effect of ouabain on Na(+) and K(+) concentrations in slices of lactating rabbit mammary-gland tissue incubated for 1h at 37 degrees C in Krebs bicarbonate buffer was also studied. 2. Prolactin increased the concentration of K(+) in mammary explants, an effect prevented by ouabain. In slices of lactating tissue, there was a linear relationship between the log dose of ouabain (from 0.1 to 10mum) and increased Na(+) and decreased K(+) concentrations in the tissue. 3. Ouabain at concentrations up to 1mum did not affect the rate of synthesis of RNA, protein or fatty acids by explants cultured with insulin and corticosterone. By contrast, the stimulatory effect of prolactin on protein synthesis was diminished and the induction of medium-chain fatty acid synthesis by prolactin was almost abolished. RNA synthesis was unaffected. Histological examination showed no tissue damage by 1mum-ouabain. 4. Explants cultured in the presence of 2mum-ouabain for 24h retained their ability to respond to prolactin when the ouabain was removed from the culture medium. Between 24 and 48h they showed responses to prolactin of a magnitude similar to those of explants never exposed to ouabain. 5. These results show that a fully functional Na(+)/K(+)-dependent adenosine triphosphatase system is necessary for prolactin to promote secretory activity in rabbit mammary gland.
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PMID:Inhibition by low concentrations of ouabain of prolactin-induced lactogenesis in rabbit mammary-gland explants. 56 78


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