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)

To study acidification mechanisms in the distal nephron, microsomes were prepared from rat renal medulla by differential centrifugation. Microsomes were enriched in the enzyme marker gamma-glutamyl transferase and contained an ATP-dependent proton pump, as evidenced by ATP-dependent, 3,3',4',5-tetrachlorosalicylanilide-reversible quenching of acridine orange fluorescence. Acidification was vanadate-insensitive, but was completely inhibited by micromolar N-ethylmaleimide. Maximal acidification was achieved in the presence of halide (Cl-, Br-) only and was not attainable with potassium-valinomycin diffusion potentials without halide ion. Microsomal ATPase activity was neither chloride- nor N-ethylmaleimide-sensitive. A chloride conductance was observed only with vesicles which had undergone ATP-dependent acidification. An ATP-dependent, N-ethylmaleimide-inhibitable, 3,3',4',5-tetrachlorosalicylanilide-reversible, and chloride-attenuated quench of bis(1,3-dibutylbarbituric acid-(5] pentamethinoxonol fluorescence was seen, consistent with net transfer of positive charge into the vesicles. Nonetheless, positive intravesicular potentials increased the ATP-dependent initial acidification rate, perhaps by increasing availability of chloride ion to the transport site. Our results are consistent with an electrogenic, ATP-dependent proton pump regulated by a voltage-sensitive chloride site.
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PMID:Characterization of an electrogenic ATP and chloride-dependent proton translocating pump from rat renal medulla. 299 42

A study was made of the dependence of ATP hydrolysis intensity upon different ratios of sodium and potassium ions in plasma membrane of L cells and of cells of clone Lebr 625, sensitive and resistant to ethidium bromide, and of the distribution of cells according to cell cycle phases in dense and sparse cultures. In dense cultures, the cell growth is arrested on G1 phase, the hydrolytic activity of (Na+ + K+)-ATPase decreases, and the Na+, K+ ratio for maximum activity of (Na+ + K+)-ATPase changes. The higher proliferative activity of Lebr 625 cells in dense culture corresponds to the higher hydrolytic activity of (Na+ + K+)-ATPase.
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PMID:[Na,K-ATPase function and cellular proliferative activity in culture]. 300 34

In HeLa cells, stable mutants which are between 25-to about 200-fold resistant to the cardiac glycoside derivative SC4453 (a digoxin analog which contains a pyridazine ring in place of a lactone ring in the C-17 position) have been isolated after a single step selection in the presence of the drug. Based on their cross-resistance pattern towards various cardiac glycosides, the mutants resistant to SC4453 (SCR mutants) appear to be of two different kinds and they differ from the two classes of ouabain-resistant mutants described previously (Gupta, R. S., and Chopra, A. (1985) J. Biol. Chem. 260, 6843-6850). One type of SCR mutants (designated as group C) exhibit a high degree of cross-resistance to all cardiac glycosides and their genins (viz. ouabain, digitoxin, digoxin, digoxigenin, convallatoxin, gitoxin, strophanthidin, and bufalin). In contrast, the second type of SCR mutant (group D) exhibit considerable resistance to only SC4453, digoxin, and digoxigenin, but showed very little or no cross-resistance to the other cardiac glycosides examined. The cross-resistance of the mutants towards cardiac glycosides was highly specific as they exhibited no cross-resistance towards a large number of other structurally and functionally related compounds (viz. ethacrynic acid, sanguinarine nitrate, penicillic acid, methyl quinolizinum bromide, 5,5'-diphenylhydantoin, deoxycorticosterone, vanadium pentoxide, and adriamycin). The cellular uptake of 86Rb in the mutant cells was found to be resistant to specific cardiac glycosides. Studies on the sensitivity of plasma membrane Na+/K+-ATPase to cardiac glycosides show that about 10-15% of the enzymic activity in the mutant cells was highly resistant to inhibition by the specific drugs to which the mutants exhibit increased resistance. Very interestingly, when the mutant cells are grown in cardiac glycoside-containing medium, the resistant form of the enzyme accounts for about 50-60% of the total enzyme. These results show that both classes of SCR mutants are affected in Na+/K+-ATPase and that the amount of the resistant enzyme in the mutant cells is regulated in response to cardiac glycosides.
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PMID:Biochemical and cross-resistance studies with HeLa cell mutants resistant to cardiac glycoside SC4453. Regulation of the resistant form of Na+/K+-ATPase in the mutant cells. 300 91

A study was made of the ouabain effect (10(-3] on cell proliferation and the dependence of ATP hydrolysis on Na/K-concentration in homogenates of mouse hepatoma (XXIIa) and of L-cells, both sensitive and resistant to etidium bromide. Na+, K+-ATPase activity was found in homogenates of cells from sparse cultures in the presence of ouabain, the activity being stimulated by the Na/K-ratio pecular for the maximum enzymatic activity in cells from the dense cultures. The effect of ouabain on the cell proliferation is similar to the effect of transition of sparse cultures to dense ones.
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PMID:[Effect of ouabain on the Na+, K+-ATPase function and multiplication of transformed cells in culture]. 300 84

The relative toxicity of numerous cardiotonic steroids (viz. ouabain, digitoxin, digoxin, convallatoxin, SC4453, bufalin, gitaloxin, digoxigenin, actodigin, oleandrin, digitoxigenin, gitoxin, strophanthidin, gitoxigenin, lanatosides A, B and C, alpha- and beta-acetyl digoxin, alpha- and beta-methyl digoxin) and related compounds towards a number of independent cell lines established from human, monkey, mouse, Syrian hamster, and Chinese hamster have been determined. All cardiac glycosides and their genins, as well as the cardiotonic alkaloid cassaine, exhibited greater than 100-fold higher toxicity towards cultured human and monkey cells in comparison to the cell lines of mouse, Syrian hamster, and Chinese hamster origins. These differences are species-related as all cell lines (both normal as well as transformed) from any one species, as well as cells from the closely related species (e.g., man and monkey or mouse, Chinese hamster, and Syrian hamster), showed similar sensitivity towards these drugs. The failure to see any significant differences in cellular toxicity for a larger number of other compounds which either bear limited structural resemblance to cardiac glycosides (viz. estradiol 17-beta-acetate, testosterone propionate, 21-acetoxy pregnenolone, beta-estradiol, digitonin, tigogenin, and tomatine) or interact with the Na+/K+ ATPase in a different manner (viz. veratridine, sanguinarine nitrate, penicillic acid, vanadium pentoxide, harmaline-HCI,5,5'-diphenyl hydantoin, quindonium bromide, and methyl quinolizinum bromide) provides strong evidence that the observed species-related differences are highly specific for cardiotonic steroids. Studies on the binding of [3H]ouabain show that, in comparison to human and monkey cell lines, no significant binding of the drug is observed in cells derived from the resistant species (i.e., mouse and Chinese hamster). The Na+/K+ ATPase from cells of the resistant species is inhibited at much higher concentrations of ouabain and digitoxin in comparison to the enzyme from human cells, and a good correlation is observed between these concentrations and those reported for inhibition of the enzyme from isolated heart muscles of the same species. These results provide strong evidence that the species-related differences in sensitivity to digitalis have a cellular basis and that the cultured cells from various mammalian species provide a useful model system for investigating the mechanism of action of cardiac glycosides.
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PMID:Cellular basis for the species differences in sensitivity to cardiac glycosides (digitalis). 300 93

Sodium ion and potassium ion activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-[3H]ethylmaleimide while it was poised in three different conformations, ostensibly E2-P, E2, and E1, respectively. These assignments were made from a consideration of the particular concentrations of ligands in the respective alkylation mixtures. After a 30-min reaction, the remaining enzymatic activity was found to vary among these three different samples from 90 to 30% of that of unalkylated controls. In all cases, the alpha polypeptide was purified and subjected to digestion with cyanogen bromide, and in each digest the same two distinct radioactive peptides were identified and purified by gel filtration on a column of Sephadex LH-60. The incorporation of N-[3H]ethylmaleimide into one of these two peptides correlated closely with enzymatic inactivation, while the incorporation into the other was most extensive when the portion of the active site to which ATP binds was unoccupied. Alkylation of the residue within the latter peptide, however, does not result in inactivation of the enzyme. Both peptides were further purified by high-pressure liquid chromatography, and their amino-terminal sequences were determined by manual dansyl Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluoresceinyl 5'-isothiocyanate [Farley, R. A., Tran, C. M., Carilli, C. T., Hawke, D., & Shively, J. E. (1984) J. Biol. Chem. 259, 9532-9535].
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PMID:Purification of labeled cyanogen bromide peptides of the alpha polypeptide from sodium ion and potassium ion activated adenosinetriphosphatase modified with N-[3H]ethylmaleimide. 301 3

There are at least four forms of DNA-dependent ATPase in mouse FM3A cells [Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., & Yamada, M. (1984) Biochemistry 23, 529-533]. One of these, ATPase B, has been purified and characterized in detail. During the purification of the enzyme, we encountered the difficulties that the enzyme could not be recovered well from the single-stranded DNA-cellulose column and that the enzyme activity was distributed very broadly. The problems were resolved by the addition of ATP in the elution buffer. The ATPase has a sedimentation coefficient of 5.5 S in both high salt and low salt. The enzyme hydrolyzes rNTPs and dATP, but ATP and dATP are preferred substrates. Adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), 5'-adenylyl methylenediphosphate (AMP-PCP), and 5'-adenylyl imidodiphosphate (AMP-PNP) inhibit the enzyme activity. The enzyme is insensitive to ouabain, oligomycin, novobiocin, and ethidium bromide. A divalent cation (Mg2+ congruent to Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Native DNA was little effective with an efficiency of 29% of that obtained with heat-denatured DNA. In addition, the enzyme showed almost no activity with poly(dA).poly(dT) although it showed very high activity with the noncomplementary combination of poly(dT) and poly(dC), suggesting that ATPase B requires single-stranded DNA for activity. ATP altered the affinity of ATPase B for single-stranded DNA. The interaction of the enzyme with DNA was studied by Sephadex G-200 gel filtration assay.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of a deoxyribonucleic acid dependent adenosinetriphosphatase from mouse FM3A cells: effects of ribonucleoside triphosphates on the interaction of the enzyme with single-stranded DNA. 301 1

Membrane fractions were isolated from the rectal gland of Squalus acanthias using differential centrifugation and a sucrose gradient run in the presence of 1 M KBr. Using the basolateral membrane marker Na+-K+-ATPase, we obtained a sixfold purification with the most highly purified fraction from the gradient (sp act = 336 +/- 37 mumol X mg protein-1 X h-1). Electrogenic Br- transport was used as a marker activity of the apical membrane, which enabled the identification and purification of a membrane fraction that is highly resolved from the basolateral membrane. The most active fraction was purified approximately 50-fold compared with the crude homogenate. In this fraction, the specific activity of electrogenic anion transport was 296 +/- 87 nmol X mg protein-1 X min-1, whereas the ATPase was only 17.6 +/- 5.7 mumol X mg protein-1 X h-1, representing about a 4-5% contamination of the apical fraction with the basolateral membrane.
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PMID:Resolution of apical from basolateral membrane of shark rectal gland. 302 92

Rat liver multivesicular bodies (MVB), as well as other hepatic subcellular organelles, are acidified by an electrogenic ATP-dependent proton pump that requires Cl- for maximal acidification (Van Dyke, R. W., Hornick, C. A., Belcher, J., Scharschmidt, B. F., and Havel, R.J. (1985) J. Biol. Chem. 260, 11021-11026), suggesting that Cl- serves as a permeable charge-compensating anion. However, we have observed that NO3- is unable to substitute for Cl-. This study was undertaken therefore to examine more closely the effects of Cl- on MVB acidification and to determine whether NO3- and other anions interact with the proton pump. ATP-dependent vesicle acidification and membrane potential (psi) were measured using the fluorescent dyes acridine orange and Oxonol V (bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol), respectively. Cl- both stimulated acidification (Km = 23.2 +/- 4.2 mM) and decreased psi (IC50 = 3.4 +/- 0.6 mM) in a concentration-dependent, nonlinear fashion. In the presence of saturating Cl- (100 mM), however, NO3- (shown to be more permeable than Cl-) and the impermeant anions SO4(2-) and PO4(2-), inhibited both ATP-dependent acidification and psi in a concentration-dependent manner. Other anions, including gluconate and HCO3-, had no effect. The inhibitory effect of NO3- was reversible. Neither SO4(2-) nor PO4(2-) appeared to block Cl- movement across the vesicle membrane as assessed by the ability of Cl- to decrease an established psi. In additional experiments, the effects of anions on relaxation of a previously established pH gradient were measured. Compared to Cl- or gluconate, NO3- had no significant effect on pH gradient relaxation, even when MVB were preloaded with NO3-, indicating that rapid cycling of NO3-/HNO3 across the MVB membrane does not occur. The organic nitrate, isosorbide dinitrate, also inhibited both acidification and psi and, similar to NO3-, had no effect on pH gradient relaxation. By contrast, NO2- potently inhibited both MVB acidification and psi but also rapidly relaxed a pre-established pH gradient, suggesting that NO2- increases MVB membrane proton permeability. Finally, MVB exhibited N-ethylmaleimide-sensitive ATPase activity that was inhibited 23.9% by NO3- (100 mM). In conclusion, although MVB are permeable to a variety of anions (Cl-, Br-, NO3-, NO2-), only Cl- and Br- support maximal rates of acidification by the proton pump.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Anion inhibition of the proton pump in rat liver multivesicular bodies. 302 35

The effect of nolinium bromide [2-(3,4-dichlorophenylamino)quinolizium bromide], which acts as a K+ antagonist in the gastric H+ +K+-dependent ATPase reaction, was investigated at the level of 32P-labelled intermediates of the gastric ATPase reaction. A concentration-dependent effect of nolinium bromide was observed on the concentrations of phosphorylated intermediates. At low (up to 50 microM) concentrations the drug did not interfere with the concentrations of intermediates but exhibited a competition with K+ at the level of both 32P-labelled intermediates and hydrolysis of ATP at pH 7.0. Similar competition was noted in the H+ +K+-dependent ATPase reaction. Low nolinium bromide concentrations also drastically slowed the enzyme turnover. The concentrations of the intermediates were lowered appreciably between 50 microM- and 100 microM-nolinium bromide without affecting the ATP hydrolysis, and the effects were independent of pH. Similar to the effects at pH 7.0, the drug also exhibited competition with K+ in lowering the E approximately P concentration at pH 5.0. A dramatic effect of pH on the K+-sensitivity as well as on turnover of the 32P-labelled intermediates was observed. Although the concentrations of intermediates remained nearly unaltered at various pH values, the K+-stimulated hydrolysis of ATP showed an optimum at pH 7.0 with sharp declines at pH 5 and 8. The data suggest a critical involvement of H+ in the conversion of the K+-insensitive E1 approximately P into the K+-sensitive E2 approximately P form of the enzyme. Nolinium bromide appears to function as a K+ analogue and seems to block the entry of K+ at the K X E2 step, thereby interfering with the enzyme turnover.
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PMID:Reaction mechanism of the gastric H+ +K+-dependent ATPase. Effects of inhibitor and pH. 303 53


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