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)

Vacuolar H(+)-ATPase was isolated from highly purified bovine kidney brush border, using a previously described immunoaffinity method. The affinity purified enzyme had reconstitutively active ATP-induced acidification that was inhibited by N-ethylmaleimide. The brush border H(+)-ATPase had a single pH optimum of 7.3, and a single Km for ATP of 360 microM. The enzyme showed no lipid activation; it had a substrate preference of ATP greater than ITP greater than UTP greater than GTP much greater than CTP, with an ATP:GTP selectivity of 1.69. The brush border H(+)-ATPase required no monovalent anion or cation for activity and was inhibited by the oxyanions NO3(-1) much greater than SO4(-2); sulfite stimulated activity at low concentrations and inhibited at higher concentrations. The inhibition produced by nitrate could not be attributed to dissociation of subunits from the enzyme. The divalent or trivalent cation preference was Mn+2 much greater than Mg+2 much greater than Co+2 greater than Al+3 greater than Ca+2 much greater than Ba+2,Sr+2; 1 mM Zn+2 inhibited the enzyme completely, but Cu+2 inhibited only 49% of activity at concentrations up to 5 mM. Sodium dodecyl sulfate-polyacrylamide gels of the brush border H(+)-ATPase showed subunits at Mr 70,000, a doublet at 56,000, 45,000, 42,000, 38,000, 33,000, 31,000, 15,000, 14,000, and 12,000. On two-dimensional gels, the pl value for the Mr 70,000 subunit was 6.3, for the Mr 56,000 was 6.4, and for the Mr 31,000 was 7.5-8.5, and microheterogeneity was observed in the Mr 56,000 and 31,000 subunits. A comparison of kidney cortex brush border H(+)-ATPase with kidney cortex microsomal H(+)-ATPase revealed differences in pH optimum, Km for ATP, lipid dependence, substrate preference, divalent ion preference, copper sensitivity, and in microheterogeneity of the Mr 56,000 and 31,000 subunits, providing evidence that different functional and structural classes of vacuolar H(+)-ATPase are segregated to specific membrane compartments.
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PMID:Isolation and properties of bovine kidney brush border vacuolar H(+)-ATPase. A proton pump with enzymatic and structural differences from kidney microsomal H(+)-ATPase. 214 91

A well-characterized chicken osteoclast plasma membrane vesicle preparation manifested Mg2(+)-dependent ATP hydrolyzing activity of 0.213 mumol inorganic phosphate released per mg protein per minute (n = 7). The Mg2+ dependence showed a high-affinity component with a KMg of 1.293 microM and Vmax of 0.063 mumol Pi per mg protein per minute, and a low-affinity component with a KMg of 297.6 microM and a Vmax of 0.232 mumol Pi per mg protein per minute. The Mg2(+)-ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (DCCD, 0.2 mM, 50.7%), N-ethylmaleimide (0.5 mM, 34.6%), nolinium bromide (1 mM, 29.9%), 4,4'-diisothiocyano-2,2'-stilbene sulfonic acid (DIDS, 1 mM, 45.1%), and p-chloromercuribenzoic acid (PCMB, 0.1 mM, 33.8%). Sodium orthovanadate (Na3 VO4) at 1 microM had no effect but caused 29.5% inhibition at 1 mM. Na+ could substitute for K+ without loss of activity, NO3- caused 19.5% inhibition when substituted for Cl-, and acetate replacement of Cl- resulted in 36.4% stimulation of Mg2(+)-ATPase. ATP, GTP, ITP, CTP, and ADP were all hydrolyzed effectively. DCCD (0.2 mM), NEM (0.5 mM), nolinium bromide (1 mM), and DIDS (50 microM) almost completely abolished proton transport as measured spectrofluorometrically by acridine orange quenching. Na3 VO4 (1 mM) had no effect, and duramycin (80 micrograms/ml) inhibited transport 52.7%. K+ replacement of Na+ caused a 79.2% increase in initial proton transport rate. NO3- and acetate substitution of Cl- resulted in a 46.1 and 55.7% decrease in transport, respectively. ATP supports transport far more effectively than the other nucleotides tested. ADP was ineffective. Experiments using the potassium ionophore, valinomycin, indicated that the proton pump functions electrogenically, with Cl- most likely cotransported by an anion transporter. The proton pump also seems to have at least one anion-sensitive site, elucidated by experiments in the presence of NO3- and Cl-.
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PMID:Biochemical characterization of an electrogenic vacuolar proton pump in purified chicken osteoclast plasma membrane vesicles. 216 21

Batch cultures of Pseudomonas mendocina, grown in rich medium with glucose excess, showed metabolic differences dependent upon whether the growth conditions were aerobic or anaerobic, with or without added electron acceptor. Under anaerobic conditions in the absence of nitrate, P. mendocina reached the stationary phase of growth after 2 or 3 days, followed by a stationary phase of 4 to 5 days. Under these conditions, a mixed-type fermentative metabolism (formic, lactic, and acetic acids) appeared. A fivefold-higher specific rate of glucose consumption and eightfold-higher production of organic acids, compared with aerobic cultures, were shown by this microorganism growing anaerobically in the absence of exogenous electron acceptors. The gradients of organic acid produced by P. mendocina under these conditions reached a maximum (lactate, 180 mV; formate, 150 mV; acetate, 215 mV) between days 2 and 3 of culture. The proton motive force (delta p) decreased during growth from -254 to -71 mV. The intracellular pH remained alkaline during the culture, reaching a steady-state value of 7.9. The gradients of organic acids apparently contributed to the generation of a delta p, which, according to the Energy Recycling Model (P. A. M. Michels, J. P. J. Michels, J. Boonstra, and W. N. Konings, FEMS Microbiol. Lett. 5:357-364, 1979), would produce an average energy gain of 1 or 1.5 mol of ATP equivalents per mol of glucose consumed with H+/ATP stoichiometry of 3 or 2, respectively. Low YATP and Yglucose values were observed, suggesting that an uncoupled metabolism exists; i.e., ATP produced by catabolic processes is not directly used for biomass synthesis. This metabolic uncoupling could be induced at least in part by organic acids and the ATP wastage could be induced by a membrane-bound ATPase involved in intracellular pH regulation.
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PMID:Proton motive force, energy recycling by end product excretion, and metabolic uncoupling during anaerobic growth of Pseudomonas mendocina. 225 45

The apical membrane of the rabbit corneal endothelium contains a potassium-selective ionic channel. In patch-clamp recordings, the probability of finding the channel in the open state (Po) depends on the presence of either HCO3- or Cl- in the bathing medium. In a methane sulfonate-containing bath, Po is less than 0.05 at all physiologically relevant transmembrane voltages. With 0 mM [HCO3-]o at +60 mV, Po was 0.085 and increased to 0.40 when [HCO3-]o was 15 mM. With 4 mM [Cl-]o at +60 mV, Po was 0.083 and with 150 mM Cl-, Po increased to 0.36. Low Po's are also found when propionate, sulphate, bromide, and nitrate are the primary bath anions. The mechanism of action of the anion-stimulated K+ channel gating is not yet known, but a direct action of pH seems unlikely. The alkalinization of cytoplasm associated with the addition of 10 mM (NH4)2SO4 to the bath and the acidification accompanying its removal do not result in channel activation nor does the use of Nigericin to equilibrate intracellular pH with that of the bath over the pH range of 6.8 to 7.8. Channel gating also is not affected by bathing the internal surface of the patch with cAMP, cGMP, GTP-gamma-s, Mg2+ or ATP. Blockers of Na/H+ exchange, Na(+)-HCO3- cotransport, Na(+)-K+ ATPase and carbonic anhydrase do not block the HCO3- stimulation of Po. Several of the properties of the channel could explain some of the previously reported voltage changes that occur in corneal endothelial cells stimulated by extracellular anions.
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PMID:Potassium channel in rabbit corneal endothelium activated by external anions. 231 91

The elasmobranch rectal gland has served as a useful model to study features of Na-K-Cl cotransport that are common to many chloride-transporting epithelia. These include: (1) dependence on a Na+ gradient created by Na-K-ATPase; (2) high intracellular Cl- concentration; (3) characteristic inhibitor profile including inhibition by loop diuretics and barium but not by amiloride, SITS, DIDS, or carbonic anhydrase inhibitors; and (4) remarkable energy efficiency of transepithelial transport (25-30 NaCl/l 02). The mechanism by which this is accomplished is clarified by kinetic analysis of experiments with isolated perfused rectal glands of Squalus acanthias in which perfusate concentrations of Na and Cl are systematically varied. These show a Hill coefficient of one for Na+ and two for Cl-, suggesting that one Na+, one K+, and two Cl- interact with the cotransport carrier. Nitrate can substitute for Cl- to some extent, and it itself weakly transported. The loop diuretic bumetanide behaves like a competitive inhibitor of Cl-. The teleological significance of the neutral cotransport of two Cl- with one Na+ and one K+ is that it enables transporting epithelia like the rectal gland, cornea, salivary gland, and thick ascending limb of Henle's loop to double the efficiency of their Na-K-ATPase pump.
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PMID:Na-K-Cl cotransport in chloride-transporting epithelia. 241 26

The characteristics of the H+ pump in isolated rat renal endocytotic vesicles were studied by the delta pH-sensitive dye acridine orange, the voltage-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide, and by a coupled optical ATPase assay. Intravesicular acidification depended on ATP and Mg2+ concentrations with half-maximal activations at 73 and 77 microM, respectively. CTP, GTP, UTP, and ITP partially supported acidification, but ADP and AMP did not. Ouabain, ethoxzolamide, levamisole, and vanadate did not inhibit H+ uptake into endocytotic vesicles. Oligomycin inhibited partially. Depending on concentration and preincubation time, Dio-9, filipin, N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD) inhibited H+ uptake completely. Filipin and, partially, DCCD acted nonspecifically by dissipating pH gradients. A specific cation was not required for the H+ pump; Zn2+ inhibited. Compared with mannitol, ATP-driven H+ uptake was stimulated by SCN- greater than Cl- greater than Br- greater than I- much greater than HPO4(2-) = gluconate = HCO3- = F-, but not by SO4(2-), NO3-, CH3COO-, S2O3(2-), and S4O6(2-). Chloride stimulated H+ uptake from the outside of the vesicles with an apparent Km of 27 mM. In the absence of Cl-, ATP-driven proton uptake was increased by intravesicular K+ and valinomycin, suggesting that the pump is electrogenic. The electrogenicity, however, could not be demonstrated with voltage-sensitive dyes. The vesicle membrane contains no significant K+ and Cl- conductances; only a conductance for H+ was found. The vesicles exhibited an ouabain-, oligomycin-, and vanadate-insensitive ATPase activity that was inhibited by DCCD and NEM. Our data indicate the presence of an electrogenic H+ pump in endocytotic vesicles from rat renal proximal tubules with similar characteristics as H+ pumps present in various intracellular (nonmitochondrial) membranes.
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PMID:Characteristics of the proton pump in rat renal cortical endocytotic vesicles. 242 58

Regulation of steady-state free Ca2+ concentration at rest and at stimulation have been studied in isolated permeabilized pancreatic acinar cells by measuring the free Ca2+ concentration of the surrounding incubation medium with a Ca2+-specific electrode. Ca2+ transport mechanisms have been further characterized in subcellular membrane fractions by measuring 45Ca2+ uptake into membrane vesicles and protein phosphorylation using polyacrylamide gel electrophoresis. (a) In permeabilized isolated acinar cells from exocrine glands, inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from endoplasmic reticulum. (b) Secretagogue-induced Ca2+ release from permeabilized cells is accompanied by increased production of IP3. At rest, steady-state free Ca2+ concentration is regulated at 4 X 10(-7) mol/L by the rough endoplasmic reticulum (RER). Ca2+ uptake into this pool is promoted by a (Ca2+ + Mg2+)-ATPase, and is dependent on cations and anions in the incubation medium in the order K+ greater than Na+ greater than Li+ greater than choline+ and Cl- greater than Br- greater than SO4(2-) = NO3- greater than I- greater than cyclamate- greater than SCN-, respectively. Similarly, Ca2+-stimulated 32P incorporation from [gamma 32P]ATP into a 130 kD protein intermediate of (Ca2+ + Mg2+)-ATPase, as well as 32P liberation, indicating (Ca2+ + Mg2+)-ATPase activity, are cation dependent. While 32P incorporation is highest in the presence of choline, 32P liberation is higher with K+, as compared with Na+ or choline, indicating that K+ ions facilitate dephosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Intracellular messengers in stimulus-secretion coupling of pancreatic acinar cells. 243 35

Modification of our previous procedure for the isolation of microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue allowed the recovery of sealed membrane vesicles displaying proton transport activity sensitive to both nitrate and orthovanadate. In the absence of a high salt concentration in the homogenization medium, contributions of nitrate-sensitive (tonoplast) and vanadate-sensitive (plasma membrane) proton transport were roughly equal. The addition of 0.25 M KCl to the homogenization medium increased the relative amount of nitrate-inhibited proton transport activity while the addition of 0.25 M KI resulted in proton pumping vesicles displaying inhibition by vanadate but stimulation by nitrate. These effects appeared to result from selective sealing of either plasma membrane or tonoplast membrane vesicles during homogenization in the presence of the two salts. Following centrifugation on linear sucrose gradients it was shown that the nitrate-sensitive, proton-transporting vesicles banded at low density and comigrated with nitrate-sensitive ATPase activity while the vanadate-sensitive, proton-transporting vesicles banded at a much higher density and comigrated with vanadate-sensitive ATPase. The properties of the vanadate-sensitive proton pumping vesicles were further characterized in microsomal membrane fractions produced by homogenization in the presence of 0.25 M KI and centrifugation on discontinuous sucrose density gradients. Proton transport was substrate specific for ATP, displayed a sharp pH optimum at 6.5, and was insensitive to azide but inhibited by N'-N-dicyclohexylcarbodiimide, diethylstilbestrol, and fluoride. The Km of proton transport for Mg:ATP was 0.67 mM and the K0.5 for vanadate inhibition was at about 50 microM. These properties are identical to those displayed by the plasma membrane ATPase and confirm a plasma membrane origin for the vesicles.
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PMID:Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue. 243 61

The clathrin-coated vesicle proton-translocating complex is composed of a maximum of eight major polypeptides. Of these potential subunits, only the 17-kDa component, which is a proton pore, has been defined functionally (Sun, S.Z., Xie, X. S., and Stone, D. K. (1987) J. Biol. Chem. 262, 14790-14794). ATPase-and proton-pumping activities of the 200-fold purified proton-translocating complex are supported by Mg2+, whereas Ca2+ will only activate ATP hydrolysis. Like Mg2+-activated ATPase activity, Ca2+-supported ATP hydrolysis is inhibited by N-ethylmaleimide, NO3-, and an inhibitory antibody and is stimulated by Cl- and phosphatidylserine. Thus, Ca2+ prevents coupling of ATPase activity to vectoral proton movement, and Ca2+-activated ATPase activity is a partial reaction useful for analyzing the subunit structure required for ATP hydrolysis. The 530-kDa holoenzyme was dissociated with 3 M urea and subcomplexes, and isolated subunits were partially resolved by glycerol gradient centrifugation. No combination of these components yielded Mg2+-activated ATPase or proton pumping. Ca2+-activated ATP hydrolysis was not catalyzed by a subcomplex containing the 70- and 58-kDa subunits but was restored by recombination of the 70-, 58-, 40-, and 33-kDa polypeptides, indicating that these are subunits of the clathrin-coated vesicle proton pump which are necessary for ATP hydrolysis.
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PMID:Partial resolution and reconstitution of the subunits of the clathrin-coated vesicle proton ATPase responsible for Ca2+-activated ATP hydrolysis. 245 21

The initial mechanisms of injury to the proximal tubule following exposure to nephrotoxic heavy metals are not well established. We studied the immediate effects of silver (Ag+) on K+ transport and respiration with extracellular K+ and O2 electrodes in suspensions of renal cortical tubules. Addition of silver nitrate (AgNO3) to tubules suspended in bicarbonate Ringer's solution caused a rapid, dose-dependent net K+ efflux (Km = 10(-4) M, Vmax = 379 nmol K+/min/mg protein) which was not inhibited by furosemide, barium chloride, quinine, tetraethylammonium, or tolbutamide. An increase in the ouabain-sensitive oxygen consumption rate (QO2) (13.9 +/- 1.1 to 25.7 +/- 4.4 nmol O2/min/mg, P less than 0.001), was observed 19 sec after the K+ efflux induced by AgNO3 (10(-4) M), suggesting a delayed increase in Na+ entry into the cell. Ouabain-insensitive QO2, nystatin-stimulated QO2, and CCCP-uncoupled QO2 were not significantly affected, indicating preserved function of the Na+,K+-ATPase and mitochondria. External addition of the thiol reagents dithiothreitol (1 mM) and reduced glutathione (1 mM) prevented and/or immediately reversed the effects on K+ transport and QO2. We conclude that Ag+ causes early changes in the permeability of the cell membrane to K+ and then to Na+ at concentrations that do not limit Na+,K+-ATPase activity or mitochondrial function. These alterations are likely the result of a reversible interaction of Ag+ with sulfhydryl groups of cell membrane proteins and may represent initial cytotoxic effects common to other sulfhydryl-reactive heavy metals on the proximal tubule.
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PMID:Silver ion (Ag+)-induced increases in cell membrane K+ and Na+ permeability in the renal proximal tubule: reversal by thiol reagents. 245 93


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