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 binding of N-acetyl-L-glutamate, the physiological allosteric activator, to rat liver carbamoyl-phosphate synthetase (ammonia) was studied by techniques of rate of dialysis and of ultracentrifugation in the Airfuge. There is one binding site for acetylglutamate per enzyme monomer (Mr 165 000). K+, Mg2+ (free) and ATP were required to demonstrate binding. The concentrations of ATP required indicate that binding of ATPA (the ATP molecule that yields Pi) is needed. HCO-3 was not essential, but it enhanced binding of acetylglutamate. Glycerol also favored binding. Plots of Kd values versus the reciprocal of free Mg2+ and ATP concentrations are linear and indicate that ATPA, K+ and Mg2+ bind before acetylglutamate. In the presence of these ligands and HCO-3, ammonia increased drastically the Kd value for acetylglutamate, whereas in absence of HCO-3 ammonia had little effect. This suggests that acetylglutamate dissociates with the products and explains the higher Km for acetylglutamate in the synthetase (overall) reaction than in the ATPase (partial) reaction. In the absence of ATP acetylglutamate was bound with high affinity if ADP and carbamoyl phosphate were present. ADP or carbamoyl phosphate alone did not promote substantial binding. Binding of acetylglutamate at low concentration was slow; it was accelerated at higher concentrations of the activator. Exchange of bound acetylglutamate with acetylglutamate in solution was fast. A scheme proposed earlier for allosteric activation of the enzyme [Rubio, V., Britton, H. G. and Grisolia, S. (1983) Eur. J. Biochem. (in preparation)] is refined to incorporate the new information. Binding of ATPA, K+ and Mg2+ and formation of 'active CO2' (the central complex) are greatly favored by acetylglutamate.
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PMID:Binding of N-acetyl-L-glutamate to rat liver carbamoyl phosphate synthetase (ammonia). 688 68

The P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is thought to be both an ATPase that actively exports cytotoxic drugs and a Cl- channel activated by cell swelling. The partial reversal of multidrug resistance by Cl- transport blockers suggests a possible role for Cl- in Pgp-mediated drug transport. We used multidrug-resistant Chinese hamster fibroblasts and human breast cancer cells expressing Pgp to study the roles of Cl- (and also Na+ and HCO3-/CO2) on Pgp-mediated efflux of the fluorescent dye rhodamine 123 (R123). In Pgp-expressing Chinese hamster fibroblasts, exposed to isosmotic solutions, the unidirectional efflux of R123 was not measurably changed by a approximately 60-min removal of Cl- (or by exposure to Na(+)-free, or nominally HCO3-/CO2-free medium); short term (2-3 min) ion substitutions were also ineffective. In human breast cancer cells transfected with human mdr1 cDNA, hyposmotic solutions activated a Cl- current but had no effect on the Pgp-mediated unidirectional efflux of R123. Additionally, in human breast cancer cells, the intracellular presence of R123 did not prevent activation of the Cl- current by hyposmotic solution. The lack of detectable effect of removal of Cl-, Na+, or HCO3- on Pgp-mediated R123 transport rules out direct coupling between substrate transport and transport of either of these ions by Pgp. The persistence of Pgp-mediated R123 efflux in osmotically swollen cells indicates that activation of the Pgp-associated Cl- current does not hinder the Pgp pump function. The lack of effect of R123 on swelling-activated Cl- current denotes that Pgp-mediated transport of organic substrates and Pgp-associated Cl- currents can occur at the same time in a single cell. These results underscore the dissociation between Pgp-mediated active drug transport and electrodiffusive Cl- transport.
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PMID:Relationships between rhodamine 123 transport, cell volume, and ion-channel function of P-glycoprotein. 751 Feb 82

The adenosine triphosphate (ATP) generating pathways of dog inner medullary collecting ducts (IMCD) were examined in vitro using suspensions of dog IMCD tubules incubated under aerobic and anaerobic conditions. Glucose is always the preferred substrate for this tissue, even if lactate can be oxidized under aerobic conditions. The metabolism of glucose proceeds largely towards lactate accumulation in the presence or absence of oxygen. Glycogen is also consumed and more markedly so during anoxia. The pentose shunt represents a minor pathway for glucose metabolism in this tissue. Under aerobic conditions, the net oxidation of glucose to CO2 contributes significantly to the cell energetics, mitochondrial and cytoplasmic mechanisms sharing equally the ATP synthesis. In the absence of oxygen, only the cytoplasmic routes of ATP synthesis are used, but the apparent ATP turnover is markedly reduced. A marked inhibition of the activity of the Na-K-ATPase during anoxia explains this observation. The utilization of glucose for osmolyte synthesis is a minor process and appears to be suppressed under anaerobic conditions. It is concluded that the ATP turnover is low in dog IMCD cells as compared with that of other nephron segments and is largely dependent upon glucose availability under aerobic or anaerobic conditions.
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PMID:Glucose metabolism in dog inner medullary collecting ducts. 752 74

Three different mechanisms interact to control the cytosolic pH (pHi) of alveolar macrophages (M phi), namely, plasmalemmal vacuolar-type H(+)-ATPase (V-ATPase), Na+/H+ exchange, and Na(+)-independent HCO3-/Cl- exchange. To investigate the activity of plasmalemmal V-ATPase in alveolar M phi, we developed a nonlinear mathematical model of pHi regulation that incorporates the biophysical determinants of pHi and the fluxes of individual acid-base equivalents. The model was used to analyze the acid-base responses of rabbit alveolar M phi to a weak acid (propionic acid) under conditions that favored V-ATPase-mediated effects (presence of 1 mM amiloride and nominal absence of CO2). The pHi was measured using the fluorescent probe, 2',7'-biscarboxethyl-5,6-carboxyfluorescein. M phi exposure to propionic acid caused a rapid fall in pHi. Recovery of pHi after acid loading varied directly with the magnitude of the acid load. Mathematical analyses showed that pHi recovery was hindered by persistent influx of propionic acid driven, in turn, by transporter-mediated H+ extrusion and propionate efflux. Eventually, a new steady state was established in which propionate and H+ were cycled out of the M phi and propionic acid was recycled into the cell. As a consequence, model predictions of the rate of V-ATPase-mediated H+ efflux were almost twice that estimated from experimental determinations of the initial rate of pHi recovery.
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PMID:Kinetic analysis of cytosolic pH regulation in alveolar macrophages: V-ATPase-mediated responses to a weak acid. 763 10

There is now convincing evidence that in addition to the vacuolar-type H(+)-ATPase, a gastric-type H+/K(+)-ATPase participates in acidification by the distal nephron. To determine whether a similar pump exists in the turtle bladder, we examined the dependence of acid secretion on mucosal K+, and the effects of supposedly specific inhibitors of the gastric H+/K(+)-ATPase, omeprazole and SCH 28080. In CO2-stimulated bladders both drugs produced dose-dependent inhibition of electrogenic H+ secretion measured as the reverse short-circuit current (RSCC). At the highest concentrations tested, H+ secretion decreased 45 +/- 16% with mucosal and 20 +/- 7% with serosal omeprazole (P < 0.01). SCH 28080 at 400 microM produced essentially complete inhibition of H+ secretion with either mucosal or serosal application. When H+ secretion was purposefully inhibited by DIDS or an adverse mucosal pH gradient, SCH 28080 had no effect on RSCC. Removing mucosal K+ (measured K+ < 50 microM), with or without mucosal barium, had no effect on RSCC. The inhibition of RSCC by omeprazole was reversed by mercaptoethanol. Finally, HCO3 secretion, as measured by either RSCC or pH-stat titration, increased significantly in response to 400 microM SCH 28080. The results demonstrate that these compounds inhibit acid secretion by the turtle bladder but stimulate the secretion of base. In view of the total independence of acid secretion on potassium, it is unlikely that any of the bladder's acid secretion is mediated by an H+/K(+)-ATPase. The most reasonable interpretation of the data is that omeprazole and SCH 28080, previously thought to be specific inhibitors of the H+/K(+)-ATPase, also inhibit the vacuolar H(+)-ATPase of the turtle bladder. The results also indicate that HCO3 secretion by the bladder employ a different mechanism of H+ transport than is used for acid secretion; there is no simple reversal of polarity in the acid- versus base-secreting cells.
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PMID:Omeprazole and SCH 28080 inhibit acid secretion by the turtle urinary bladder. 769 39

The responses of the cytosolic pH of hepatocytes in suspension to agents affecting the activity of vacuolar adenosine triphosphatase (V-ATPase) and Na/H exchange have been studied. Changes of cytosolic pH were determined both with dual-wavelength excitation (500/440 nm) of the fluorescence of 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein and from the distribution of 14C-dimethyloxazolidinedione; both methods gave very similar results. Changes of vesicular pH were determined by comparing the fluorescence of fluorescein isothiocyanate-dextran and rhodamine B isothiocyanate-dextran taken up by endocytosis. Nitrate, which inhibits V-ATPase in isolated organelles, induced a concentration-dependent acidification of the cytosol and alkalinization of vesicles, with maximal effects at 25-37.5 mM in each case, indicating that V-ATPase contributes to removal of cytosolic protons. On continued exposure to nitrate, the acidification underwent an amiloride-inhibitable reversal. At the higher concentrations of NO3-, both cytosolic acidification and vesicular alkalinization were reduced or absent. Bafilomycin A1 caused alkalinization of vesicular pH; cytosolic acidification was not observed, possibly because of other ionic exchanges. Recovery of cytosolic pH from an acid load (2 min exposure to 5% CO2) was sensitive to both 25 mM NO3- and to ouabain. The pH dependence of the nitrate effect was tested with media of different pH; the activity was negligible at cytosolic pH 6.2 and rose to a maximum at cytosolic pH 7.3. Treatment of hepatocytes with 0.5-1.0 mM ouabain resulted in an initial alkalinization (0.5-2 min duration) of the cytosol, followed by a spontaneous reversal and, on occasion, further acidification. The alkalinization was blocked by 25 mM NO3-, but not by 25 mM gluconate. The results suggest that the cytosolic alkalinization is caused by a stimulation of H+ uptake by V-ATPase activity. We conclude that V-ATPase make an important contribution to the regulation of the cytosolic pH of hepatocytes.
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PMID:Role of vacuolar adenosine triphosphatase in the regulation of cytosolic pH in hepatocytes. 770 51

The roles of protein kinase C (PKC) in regulation of the plasmalemmal vacuolar-type H(+)-ATPase (V-ATPase) and Na(+)-H+ exchanger (NHE) of rabbit alveolar macrophages (m phi) were investigated using phorbol 12-myristate 13-acetate (PMA). At an extracellular pH (pHo) of 7.4 (nominal absence of CO2-HCO3-), PMA caused a dose-dependent increase in the rate of cellular H+ extrusion with little change in intracellular pH (pHi). PMA caused a prolonged cytosolic acidification at pHo < or = 6.8. PMA-induced changes in pHi were sensitive to bafilomycin A1, but were insensitive to amiloride. Studies of pHi recovery following intracellular acid challenge showed that both V-ATPase and the NHE were up-regulated by PMA. An inactive analog, 4 alpha-phorbol, had no detectable effects on pHi homeostasis. These data indicate that (a) PKC is involved in regulation of V-ATPase and the NHE of resident alveolar m phi and (b) V-ATPase is the predominant mechanism for pHi homeostasis in unstimulated and PMA-activated m phi.
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PMID:Effects of myristate phorbol ester on V-ATPase activity and Na(+)-H+ exchange in alveolar macrophages. 772 18

Intracellular pH (pHi) homeostasis in resident alveolar macrophages (m phi) under nominally CO2-free conditions is mediated primarily by the activity of plasmalemmal H(+)-ATPase. The m phi also possess an Na(+)-H+ exchanger (NHE) but this mechanism has no detectable role in pHi regulation in the physiologic range. To further explore the physiological significance of the NHE in this cell type, resident alveolar m phi from rabbits were subjected to a hyperosmotic challenge (approximately 620 mOsm/kg) in the nominal absence of CO2-HCO3-. Osmotic cell shrinkage was accompanied by an amiloride-sensitive increase in baseline pHi. The NHE-mediated rate of pHi recovery from intracellular acid loads also increased under hyperosmotic conditions. Cell shrinkage caused an alkaline shift in the pHi set point of the NHE without altering the exchanger's affinity for extracellular Na+. The results indicate that Na(+)-H+ exchange in resident alveolar m phi is activated by osmotic cell shrinkage and imply that the NHE may be involved in volume regulatory responses of the cell.
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PMID:Na(+)-H+ exchange in resident alveolar macrophages: activation by osmotic cell shrinkage. 772 19

Osteoclasts resorb bone by pumping of H+ into a compartment between the cell and the bone surface. Intracellular pH (pHi) homeostasis requires that this acid extrusion, mediated by a vacuolar-type H+ ATPase, be complemented by other acid-base transporters. We investigated acid-extrusion mechanisms of single, freshly isolated, neonatal rat osteoclasts. Cells adherent to glass coverslips were studied in the nominal absence of CO2/HCO3-, using the pH-sensitive dye BCECF and a digital imaging system. Initial pHi averaged 7.31 and was uniform throughout individual cells. Intrinsic buffering power (beta 1) decreased curvilinearly from approximately 25 mM at pHi = 6.4 to approximately 6.0 mM at pHi = 7.4. In all polygonally shaped osteoclasts, and approximately 60% of round osteoclasts (approximately 20% of total), pHi recovery from acid loads was mediated exclusively by Na-H exchange. In these pattern-1 cells, pHi recovery was 95% complete within 200 s, and was blocked by removing Na+, or by applying 1 mM amiloride, 50 microM ethylisopropylamiloride (EIPA), or 50 microM hexamethyleneamiloride (HMA). The apparent K1/2 for HMA ([Na+]o = 150 mM) was 49 nM, and the apparent K1/2 for Na+ was 45 mM. Na-H exchange, corrected for amiloride-insensitive fluxes, was half maximal at pHi 6.73, with an apparent Hill coefficient for intracellular H+ of 2.9. Maximal Na-H exchange averaged 741 microM/s. In the remaining approximately 40% of round osteoclasts (pattern-2 cells), pHi recovery from acid loads was brisk even in the absence of Na+ or presence of amiloride. This Na(+)-independent pHi recovery was blocked by 7-chloro-4-nitrobenz-2-oxa-1,3-diazol (NBD-Cl), a vacuolar-type H+ pump inhibitor. Corrected for NBD-Cl insensitive fluxes, H+ pump fluxes decreased approximately linearly from 96 at pHi 6.8 to 11 microM/s at pHi 7.45. In approximately 45% of pattern-2 cells, Na+ readdition elicited a further pHi recovery, suggesting that H+ pumps and Na-H exchangers can exist simultaneously. We conclude that, under the conditions of our study, most neonatal rat osteoclasts express Na-H exchangers that are probably of the ubiquitous basolateral subtype. Some cells express vacuolar-type H+ pumps in their plasma membrane, as do active osteoclasts in situ.
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PMID:Role of Na-H exchangers and vacuolar H+ pumps in intracellular pH regulation in neonatal rat osteoclasts. 776 16

The syntrophically glycolate-fermenting bacterium in the methanogenic binary coculture FlGlyM was isolated in pure culture (strain FlGlyR) with glyoxylate as sole substrate. This strain disproportionated 12 glyoxylate to 7 glycolate, 10 CO2, and 3 hydrogen. Glyoxylate was oxidized via the malyl-CoA pathway. All enzymes of this pathway, i.e. malyl-CoA lyase/malate: CoA ligase, malic enzyme, and pyruvate synthase, were demonstrated in cell-free extracts. Glycolate dehydrogenase, hydrogenase, and ATPase, as well as menaquinones as potential electron carriers, were present in the membranes. Everted membrane vesicles catalyzed hydrogen-dependent glyoxylate reduction to glycolate [86-207 nmol min-1 (mg protein)-1] coupled to ATP synthesis from ADP and Pi [38-82 nmol min-1 (mg protein)-1)]. ATP synthesis was abolished entirely by protonophores or ATPase inhibitors (up to 98 and 94% inhibition, respectively) indicating the involvement of proton-motive force in an electron transport phosphorylation driven by a new glyoxylate respiration with hydrogen as electron donor. Measured reaction rates in vesicle preparations revealed a stoichiometry of ATP formation of 0.2-0.5 ATP per glyoxylate reduced.
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PMID:Electron transport phosphorylation driven by glyoxylate respiration with hydrogen as electron donor in membrane vesicles of a glyoxylate-fermenting bacterium. 776 34


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