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)

The mechanism of the reaction catalyzed by rat liver mitochondrial carbamoyl-phosphate synthetase has been studied by using [beta-18O2]ATP and HC18O-3, monitoring the isotopic composition of adenosine triphosphate (ATP) and inorganic phosphate (Pi) by high-resolution 31P NMR spectroscopy. In the presence of both HCO3- and acetylglutamate, the enzyme catalyzes the exchange of oxygen atoms between the beta, gamma bridging and the beta nonbridging positions of ATP. Addition of NH3 stops the exchange, Pi released by the ATPase activity of the enzyme in the absence of NH3 contains one oxygen atom from HC18O3- but there is no incorporation of 18O into ATP. There is no significant incorporation of [14C]ADP or 32Pi into ATP. It is concluded that in the enzyme-ATPA.HCO30.ATPB complex formed in the presence of ATP and HCO3- there is reversible transfer of the gamma-PO3 group of ATPA (the molecule that yields Pi) to HCO3- without dissociation of products. The beta-PO3 of the enzyme-bound ADP that is formed can rotate. Virtually all of the complex appears to be in the form in which ATPA is cleaved, but in the absence of NH3, ATP is reconstituted and dissociates from the complex on at least 75% of the occasions. On the remainder, the carbonyl phosphate is cleaved in an irreversible process that yields Pi and a low-energy form of carbonic acid (probably HCO3-). NH3 reacts rapidly and irreversibly with the complex, and at saturation the rate (greater than 10 times the rate of Pi release in the absence of NH3) is sufficient to prevent dissociation of ATPA. In the absence of HCO3- an enzyme-ATPA.ATPB complex is formed, but cleavage of the bond between beta, gamma bridging oxygen and P gamma of ATPA does not occur.
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PMID:Mechanism of activation of bicarbonate ion by mitochondrial carbamoyl-phosphate synthetase: formation of enzyme-bound adenosine diphosphate from the adenosine triphosphate that yields inorganic phosphate. 626 8

It has previously been shown that there are two sites for divalent metals at the active site of kidney (Na+ + K+)-ATPase, one bound directly to the enzyme and one coordinated to the ATP substrate [Grisham, C. (1981) J. Inorg. Biochem. 14, 45; O'Connor, S., & Grisham, C. (1980) FEBS Lett. 118, 303]. The conformation of the metal-nucleotide complex has been studied by using beta, gamma-bidentate Co-(NH3)4ATP, a substitution-inert analogue of MgATP. Kinetic studies show that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the (Na+ + K+)-ATPase. The Ki values under both high- and low-affinity conditions (Ki = 10 microM and Ki = 1.6 mM, respectively) are similar to the Km values for MnATP under the same conditions (2.88 microM and 0.902 mM). From the paramagnetic effect of Mn2+ bound to the ATPase on the longitudinal relaxation rates of the phosphorus nuclei of Co(NH3)4ATP at the substrate site (at 40.5 and 145.75 MHz), Mn-P distances to all three phosphates are determined. The distances are consistent with the formation of a second sphere coordination complex on the enzyme between Mn2+ and the phosphates of Co(NH3)4ATP. In this respect, kidney (Na+ + K+)-ATPase appears to be similar to pyruvate kinase [Sloan, D., & Mildvan, A. (1976) J. Biol. Chem. 251, 2412] and phosphoribosylpyrophosphate synthetase [Granot, J., Gibson, K., Switzer, R., & Mildvan, A. (1980) J. Biol. Chem. 255, 10931]. Roles for both of the active site divalent cations are discussed.
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PMID:Phosphorus-31 nuclear magnetic resonance studies of the conformation of an adenosine 5'-triphosphate analogue at the active site of (Na+ + K+)-ATPase from kidney medulla. 629 42

The human large intestine absorbs Na+, Cl- and water from its lumen and secretes HCO-3 and some K+. The primary event in absorption is thought to be the active transport of Na+ ions out of the cell and across the baso-lateral cell membrane, by the energy requiring Na+-K+ ATPase. This leads in turn to Na+ entry into the cell via its luminal border and the creation of a potential across the mucosa which drives the transport of other ions. Cl- is coupled to HCO-3 secretion through a common carrier and K+ enters the intestinal lumen partly through an active secretory pathway. Most ions probably cross the epithelium by both transcellular and paracellular (shunt) pathways, water moving in response to solute transport. However the colon is not normally perfused by a saline-bicarbonate solution. It contains an active microflora which ferment 30 g or more of carbohydrate daily, derived from diet and intestinal secretions, with the production of at least 300 mmol of short chain fatty acids (acetic, propionic and butyric acids). About 6 g of urea is also degraded to NH3. These metabolic processes result in the generation of solutes which are then transported across the mucosa and which alter the pattern of water and electrolyte transport significantly. Short chain fatty acids are rapidly absorbed by passive diffusion as the undissociated acids, although anion transport, possibly through a paracellular route, is also feasible. Their absorption leads to the accumulation in the lumen of HCO3, a rise in pH, fall in pCO2 and stimulation of Na+ and water transport. The effect on Na+ transport is thought to indicate the presence of a Na+/H+ exchange in the cell membrane. The amounts of these organic solutes produced in the colon each day are probably greater than the total numbers of inorganic ions such as Na+, K+, Cl- and HCO-3 and as such must be taken into account in any understanding of overall transport processes in the large intestinal epithelium.
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PMID:Colonic absorption: the importance of short chain fatty acids in man. 637 78

The role of simultaneously existing ATP-binding sites in the catalytic process of Na+/K(+)-ATPase is unclear. In order to learn whether blocking the E1ATP site affects the properties of the E2ATP site, the E1ATP site was inactivated by either fluorescein 5'-isothiocyanate, the non-phosphorylating Cr(H2O)4AdoPP[CH2]P or the phosphorylating Cr(H2O)4ATP. The properties of the remaining E2ATP site were studied by measuring 'backdoor phosphorylation' in the presence of ouabain, or K(+)-activated hydrolysis of p-nitrophenyl phosphate. The involvement of the E2ATP site was further tested by the effects of Co(NH3)4ATP, a specific inactivator of this site. When the E1ATP site was inactivated by fluorescein 5'-isothiocyanate or the non-phosphorylating Cr(H2O)4AdoPP[CH2]P, backdoor phosphorylation and the activity of K(+)-activated p-nitrophenylphosphatase remained unchanged. Both processes were lost, however, when the E2ATP site was additionally inactivated by Co(NH3)4ATP. Inactivation of the E1ATP site by fluorescein 5'-isothiocyanate or Cr(H2O)4AdoPP[CH2]P decreased the affinity of the p-nitrophenylphosphatase activity of the E2ATP site for the substrate p-nitrophenyl phosphate by four times. This is consistent with a former report showing that dephosphorylation in a fluorescein 5'-isothiocyanate-inactivated Na+/K(+)-ATPase has a lowered sensitivity for ATP [Scheiner-Bobis, G., Antonipillai, J. & Farley, R. A. (1993) Biochemistry 32, 9592-9599]. Inactivation of the E1ATP site by the phosphorylating Cr(H2O)4ATP, however, led to a loss of the property of the E2ATP site to hydrolyse K(+)-dependent p-nitrophenyl phosphate and to achieve backdoor phosphorylation. Evidently, ATP sites coexist in Na+/K(+)-ATPase, and binding of ATP to one site affects the property of the other site [Scheiner-Bobis, G., Esmann, M. & Schoner, W. (1989) Eur. J. Biochem. 183, 173-178]. Although the enzyme can be phosphorylated from both ATP sites, phosphorylation of the E1ATP site excludes the phosphorylation of the E2ATP site.
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PMID:Na+/K(+)-ATPase with a blocked E1ATP site still allows backdoor phosphorylation of the E2ATP site. 755 90

The photoreduction, without reductant dithionite, of N2 to NH3 or acetylene to ethylene catalysed by nitrogenase in the presence of Mg2+. ATP, eosin and NADH in the light has been established. There is an optimum NADH concentration for each particular eosin concentration. When the ratio of the iron protein component of nitrogenase from Azotobacter vinelandii (Av2)/the molybdenum-iron protein component of nitrogenase from A. vinelandii (Av1) is equal to 3 for 4 x 10(-5) M eosin the optimum NADH concentration is 5 x 10(-4) M. The rate of photoreduction (per one electron) of acetylene or N2 under identical conditions was shown to be similar. The photoreductant-dependent ATPase activity, in the presence of a given photochemical system in the light, was revealed. Eosin is shown to be the inhibitor of the coupling site. Concentrations of 8 x 10(-6) -1 x 10(-4) M eosin do not inhibit the ATPase activity. The inhibition of substrate-reduction activity depends on the ratio of the nitrogenase components. Under conditions where the Av2/Av1 ratio is equal to 1 the rate of photochemical reduction is higher than in the presence of dithionite: the total electron flux through nitrogenase being increased 2.2-fold. We suggest that in this case the nitrogenase complex (1:1) works without dissociation.
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PMID:The photoreduction of nitrogenase. 768 Aug 58

Pathways for ammonia transport have been incorporated within a model of rat proximal tubule [A. M. Weinstein. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F784-F798, 1992]. The luminal membrane includes a Na+/NH4+ exchanger, while at the peritubular membrane there is uptake of NH4+ on the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase); both luminal and peritubular cell membranes contain conductive pathways for NH4+. The model equations have been expanded to include cellular ammoniagenesis. The principal focus of this study is the interplay of forces that can raise proximal tubule fluid total ammonia concentration 10-fold higher than in arterial plasma. Analysis of a cellular model reveals that luminal membrane Na+/NH4+ exchange, cellular production of ammonia, and peritubular membrane NH4+ uptake (via Na(+)-K(+)-ATPase or via K+ channel) all act in parallel to drive ammonia secretion. This derives from the cellular interconversion of NH4+ and NH3 and the free permeation of NH3 across cell membranes. It implies that inhibition of the luminal membrane transporter does not block the contribution of peritubular uptake to the overall active transport of ammonia. Conversely, when inhibition of the luminal membrane Na+/NH4+ entry (i.e., Na+/H+ inhibition) depresses transcellular Na+ flux, then the decrease of NH4+ flux through the peritubular Na+ pump enhances the apparent importance of the luminal membrane pathway. This analysis is confirmed in the numerical calculations and is a departure from the Ussing paradigm of series membrane Na+ transport. Although active secretion of ammonia by this tubule is substantial, the relative contribution of luminal Na+/NH4+ exchange and of peritubular uptake via the Na+ pump remains uncertain. The determination of peritubular capillary NH4+ concentration will be crucial to resolving this uncertainty, with lower concentration (i.e., closer to systemic arterial ammonia) obligating greater luminal membrane Na+/NH4+ exchange.
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PMID:Ammonia transport in a mathematical model of rat proximal tubule. 806 84

To examine functional changes of the transporters in the inner stripe of the outer medullary collecting ducts (OMCDis) by the peritubular acid-base status, in vitro microperfusion using the acetoxymethyl ester of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein was performed. Cell alkalinization systems were assessed by the recovery rate (dpHi/dt) of intracellular pH (pHi) after intracellular acid loading by NH(4+)-NH3 prepulse with bath amiloride. In alkali-loaded rabbits (0.15 M NaHCO3 drinking for 14 days), dpHi/dt showed a significant decrease (1.80 +/- 0.29 pH units/s x 10(3)) compared with either control (3.30 +/- 0.59) or acid-loaded rabbits (0.15 M NH4Cl drinking for 14 days, 3.05 +/- 0.46). The difference of dpHi/dt between control and alkali-loaded rabbits was eliminated by lumen N-ethylmaleimide (NEM), suggesting that H+ pump activity was decreased. The effect of in vitro alkali treatment (50 mM HCO3-, pH 7.7) for 3-4 h was also examined. This incubation significantly decreased the dpHi/dt (1.83 +/- 0.35) compared with the time control experiments (3.18 +/- 0.28), whereas no significant difference was seen in the presence of lumen NEM. Anion exchanger activity, as determined from the pHi changes after Cl- addition to the bath, showed no significant change with in vivo or in vitro alkali treatment. The results indicate that cell function of the OMCDis is regulated in response to the peritubular acid-base environment via changes in the H(+)-adenosinetriphosphatase.
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PMID:Effects of in vivo and in vitro alkali treatment on intracellular pH regulation of OMCDis cells. 823 55

Gastric parietal cell apical membranes must protect the cell from the extremely low pH and wide variations in osmolality of the gastric juice. To characterize the permeability properties of gastric apical membranes, we have measured passive permeabilities to water, protons, NH3, and small nonelectrolytes of membrane vesicles derived from parietal cells of fasted animals and fed animals. Both preparations are known to be highly enriched in H+/K(+)-ATPase, the enzyme responsible for acidifying the gastric contents. The preparations behaved as single populations, and their permeability properties were similar in all respects, permitting pooling of the results. This similarity suggests that insertion of tubulovesicles into the apical membrane does not change the behavior of the lipid bilayer. Osmotic water permeability (Pf) averaged (mean +/- SD) (2.8 +/- 0.3) x 10(-4) cm/s, a value 10-fold lower than that obtained in lecithin large unilamellar vesicles (LUV) and similar to that obtained in other water-tight epithelia. Similarly, ammonia permeability (PNH3) was low [(4.4 +/- 2.3) x 10(-3) cm/s] and 10 times below that of lecithin LUV. By contrast, proton permeability (PH+) was surprisingly high (0.030 +/- 0.011 cm/s) and similar to that of lecithin LUV. These results suggest that the pathway for proton permeation differs from that of water and NH3. Nonelectrolyte permeabilities were strikingly similar to those obtained in another water-tight epithelium, the toad urinary bladder. Moreover, these permeabilities followed Overton's rule in that permeability varied in accordance with the oil-water partition coefficient. We conclude that the gastric apical membrane, like that of several renal epithelia, is relatively water-tight and exhibits low permeabilities to small nonelectrolytes. These properties are likely to be essential to the ability of this membrane to perform its barrier function.
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PMID:Apical membrane of the gastric parietal cell: water, proton, and nonelectrolyte permeabilities. 838 20

The chromium complex of adenosine 5'-[beta,gamma-methylene]triphosphate, Cr(H2O)4AdoPP[CH2]P, inactivates Na+/K(+)-ATPase from pig kidney at 37 degrees C with an inactivation velocity constant of 7.1 x 10(-3) min-1 by binding to the high-affinity ATP site (E1ATP site). The dissociation constant (Kd) of the analogue at this site is 26 microM, and of ATP 0.8 microM. Inactivation of the overall reaction of Na+/K(+)-ATPase by Cr(H2O)4AdoPP[CH2]P did not alter the activities of the E2 conformational state such as K(+)-activated p-nitrophenylphosphatase, 86Rb+ occlusion and [3H]ouabain binding by the 'backdoor' phosphorylation. However, [3H]ouabain binding via the forwards reaction from E1ATP in the presence of Na+ + Mg2+ is inhibited. K(+)-activated p-nitrophenylphosphatase activity of the Cr(H2O)4AdoPP[CH2]P-inactivated enzyme decreases when an MgATP analogue, the tetraammine cobalt complex of ATP, Co(NH3)4ATP, is used additionally to inactivate the E2ATP site. The enzyme activity of K(+)-activated phosphatase is also lost if the beta,gamma-bidentate chromium(III) complex of ATP, Cr(H2O)4ATP, which may form a stable E1-chromo-phosphointermediate, is used for the inactivation of Na+/K(+)-ATPase. We conclude that the phenomenon of a blockade of the overall reaction of Na+/K(+)-ATPase by the formation of a stable E1.CrAdoPP[CH2]P complex, leading thereby to a loss of the partial activities of the E1 conformation, but not of the E2 conformation, is consistent with the postulate of an (alpha beta)2 diprotomeric nature of the sodium pump. The observation, moreover, that treatment of the sodium pump with Cr(H2O)4ATP but not with Cr(H2O)4AdoPP[CH2]P leads to an inactivation of K(+)-activated phosphatase seems to indicate that the formation of a E1-phosphointermediate affects the E2ATP site.
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PMID:Modification of the E1ATP binding site of Na+/K(+)-ATPase by the chromium complex of adenosine 5'-[beta,gamma-methylene]triphosphate blocks the overall reaction but not the partial activities of the E2 conformation. 838 35

Recent studies have indicated the presence of hydrogen-potassium-adenosinetriphosphatase (H-K-ATPase) in the collecting duct. We examined the localization of functional H-K-ATPase activity in individual cells of the outer and inner stripes of outer medullary collecting ducts (OMCDo and OMCDi). Tubules were isolated from control and K(+)-depleted rabbits and perfused in vitro. Intracellular pH (pHi) of principal cells, intercalated cells, and OMCDi cells was monitored by fluorescence ratio imaging using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). An intracellular acid load was induced by NH3/NH4 prepulse in extracellular Na(+)-, K(+)-, and HCO3(-)-free condition, and then 5 mM K+ was added to the lumen or the bath in the presence of Ba2+. Functional activity of H-K-ATPase was estimated by the difference in the rates of pHi recovery before and after K+ addition. In the control condition, luminal addition of K+ significantly increased the pHi recovery rate by 1.6 +/- 0.4 and 1.9 +/- 0.4 x 10(-3) pH units/s in intercalated calls and OMCDi cells, respectively, but not in principal cells. This K(+)-dependent pHi recovery was inhibited by 63% in intercalated cells and 74% in OMCDi cells in the presence of luminal Sch-28080 (10 microM) but was not affected in the presence of luminal bafilomycin-A1 (10 nM). K+ depletion increased the K(+)-dependent pHi recovery to 2.3-fold in intercalated cells and 2.6-fold in OMCDi cells. By contrast, K(+)-dependent pHi recovery was not detected in the basolateral membrane of any cell types in either the control or the K(+)-depleted condition. These results provide functional evidence that H-K-ATPase is distributed in the luminal membrane of intercalated cells and OMCDi cells and that this ATPase is activated by K+ depletion, suggesting the contribution of intercalated cells and OMCDi cells to K+ conservation in rabbit OMCD.
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PMID:Functional activity of H-K-ATPase in individual cells of OMCD: localization and effect of K+ depletion. 876 29


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