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)

We previously measured the amounts of Na+ and K+ ions bound to the Na+,K+-dependent ATPase [EC 3.6.1.3] purified from porcine kidney by a modified membrane filtration method [(1979) J. Biochem. 86, 509--523]. In this study, we improved the method for measuring the amount of the active site and measured the amount of Rb+ ions (a K+ congener) bound to the ATPase as well as those of Na+ and K+ ions to get more accurate information on the K+- and Na+-binding sites. The following results were obtained. Two kinds of cation-binding sites were found to exist on the ATPase molecule. One was the Na+-binding sites (3 mol per mol of active site). Na+ ions were bound to the sites cooperatively (Hill coefficient, 2.5--3), and the apparent dissociation constant was 0.20--0.32 mM. Three moles of Na+ ions bound to the sites was displaced by 1 mol of K+ ions bound to the ATPase (phi K, 24 microM). The other was the K+-binding sites (2 mol per mol of active site). Two moles of K+, Rb+, or Na+ ions was bound to the sites cooperatively (Hill coefficient, 1.5--2), and their apparent dissociation constants were 0.044, 0.024, and 2.2 mM, respectively. We measured the amounts of Na+ and Rb+ ions bound to the ATPase in the presence of 0.8 mM NaCl and 0.13 mM RbCl, and obtained unequivocal evidence for the simultaneous binding of 3 mol of Na+ ions and 2 mol of Rb+ ions per mol of active site of the ATPase.
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PMID:Binding of monovalent cations to Na+,K+-dependent ATPase purified from porcine kidney. I. Simultaneous binding of three sodium and two potassium or rubidium ions to the enzyme. 625 64

Inhibition of the Na+-K+ active transport system has been postulated to be one mechanism through which myocardial contractility is increased. Rubidium is one substance which has been shown to increase the contractility of guinea-pig atria and inhibit the activity of the isolated Na+,K+-adenosine triphosphatase of guinea-pig ventricle. A reexamination of these results confirmed the positive inotropic effect of rubidium on guinea-pig atria and demonstrated that this effect on contractility is accompanied by a decrease in both resting potential and action potential duration. However, it was also found that rubidium produced a transient negative inotropic effect in guinea-pig ventricle. The latter response was closely paralleled by a transient shortening of action potential duration. A concentration of rubidium maximally effective in decreasing contractility (2.0 mM) had no effect on the slow response action potential or contraction. RbCl (0.1 mM) had no effect on cyclic adenosine 3':5'-monophosphate levels of the ventricle or atrium. RbCl did inhibit active transport in the ventricle, as evidenced by a significant reduction in the electrogenic contribution on the active transport system to the maximal diastolic membrane potential during high-frequency drive. These results demonstrate that RbCl has different effects on the contractility of atrial and ventricular muscle. They also suggest that inhibition of the sodium pump is not necessarily accompanied by an increased force of myocardial contraction.
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PMID:Effects of rubidium on contractility and sodium pump activity in guinea-pig ventricle. 627 Mar 15

It has been shown previously that both low (less than 2 mM) and high (greater than 45 mM) concentrations of extracellular K inhibit the renin secretory rate of rat kidney slices, and that nonidentical Ca-dependent mechanisms appear to be involved. As Rb can substitute for K in many biological systems, the present experiments were designed to compare the effects of K and Rb on renin secretion of rat kidney slices. Adding either KCl or RbCl to a nominally K-free incubation medium stimulated renin secretory rate in concentration-dependent manners; secretory rate was half-maximally stimulated at approximately 1.5 mM and maximally stimulated at approximately 2-3 mM concentrations of either KCl or RbCl. Ouabain completely abolished the basal secretory rate, in either KCl- or RbCl-containing media. These results suggest that the effects of increasing KCl or RbCl in the range of 0.5-4 mM are attributable to stimulatory effects of Rb and K on Na-K-ATPase activity. Renin secretory rate was greatly inhibited by incubating kidney slices in media containing 60 mM concentrations of either KCl or RbCl. A concentration of methoxy-verapamil which completely blocked the inhibitory effects of 60 mM KCl or of 60 mM RbCl failed to antagonize the inhibitory effects of a nominally K-free medium or of media containing ouabain and either 4 mM KCl or 4 mM RbCl. Taken together with previous results, these observations suggest that Rb can substitute for K in the renin secretory process. Furthermore, they support the hypothesis that inhibitors of Na-K-ATPase, and depolarization inhibit renin secretion by Ca-dependent mechanisms which are not identical.
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PMID:Comparison of the effects of rubidium and potassium on renin secretion from rat kidney slices. 633 46

Conformational states of the Na,K-ATPase and rates of transition between these are studied using the fluorescent dye eosin as a marker for the Na+ form (E1) and occlusion of 86Rb+ as a marker for the K+ form (E2). The aim of the present paper is to propose that the E1 form of the Na,K-ATPase can be liganded with a number of Rb+ and Na+ ions and that only some of these E1 forms bind eosin and thus probably nucleotides with high affinity. Experiments are performed with Na,K-ATPase isolated from pig kidney. Binding of eosin occurs only when Na+ is present at millimolar concentrations, and the observed rate of binding is slow when Rb+ is present. The rate of eosin binding after a sudden increase in the Na+ concentration is about the same as the rate of deocclusion of Rb+, suggesting that eosin monitors the rate of the E2 to E1 transition. Titrations of eosin fluorescence with Na+ indicate that binding of more than one Na+ occurs when high-affinity eosin binding takes place. With 0.05 mM RbCl and 4 mM NaCl present, the Na,K-ATPase is a mixture of at least two enzyme species which do not bind eosin with high affinity. One species is the E2 form with Rb+ occluded, and transition of this form to E1 gives rise to a small observed rate constant for eosin binding when the Na+ concentration is suddenly increased to about 25 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Influence of Na+ on conformational states in membrane-bound renal Na,K-ATPase. 751 50

In order to assess the relationship between cytosolic [ATP] or [ATP]/[ADP] and the intracellular Na+ concentration ([Na+]i), we have used the phosphate trap 2-deoxy-D-glucose (DG) to alter the high energy phosphate levels in rat cardiomyocytes. Pyruvate-perfused rat hearts were treated with 2 mM DG in the presence of 10IU/l of insulin for 28 min, followed by perfusion with DG without insulin for 60 min. The DG + insulin treatment resulted in dramatic changes in the 31P NMR spectra: phosphocreatine (PCr) and total ATP decreased (to 15 and 35%, respectively) and deoxyglucose-6-phosphate accumulated, with little change in either inorganic phosphate or intracellular pH. These changes corresponded to a decrease in cytoplasmic [ATP] (from 7.6 to 1.8 mM), [ATP]/[ADP] (from 494 to 24) and ATP affinity [A(ATP), by 8.9 kJ/mol] and an increase in [ADP] (five-fold) and free [Mg2+] (two-fold). Subsequent perfusion with DG--insulin resulted in slow recovery of PCr, [ATP]/[ADP] and A(ATP) such that the "low energy" state lasted an additional 16 min during which ATP remained low and constant. There were no detectable changes in the intracellular Na+ content as assessed by shift reagent-aided 23Na NMR at the end of DG + insulin treatment (98 +/- 18%, 28-36 min of the protocol). In addition, there was no change in the Rb+ influx rate as measured by 87Rb NMR at the beginning of insulin washout which was achieved by replacing 20% of the KCl with RbCl ([K+] = 3.76 mM, [Rb+] = 0.94 mM). During DG + insulin treatment the pressure-rate product (PRP) decreased by half and was restored upon insulin washout to 80% of its initial value both in the presence and in the absence of the shift reagent [5 mM Dy (triethylenetetraminehexaacetate)3-]. These data imply that unfavorable thermodynamic [low A(ATP)] and kinetic (low [ATP] and [ATP]/[ADP]) conditions induced by DG treatment do not inhibit Na+, K(+)-ATPase activity. We speculate that during anoxia when changes in [ATP]/[ADP] are comparable to those induced by DG treatment, the observed increase in [Na+]i is not due to inhibition of the Na+ pump by reduced [ATP] or [ATP]/[ADP].
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PMID:Relationships between cytosolic [ATP], [ATP]/[ADP] and ionic fluxes in the perfused rat heart: A 31P, 23Na and 87Rb NMR study. 786 98

The role of the Na,K-ATPase beta-subunit in stabilization of ion-binding sites has been investigated. Treatment of the purified renal Na,K-ATPase with 0.25 M DTT at 40 degrees C for 1 h resulted in 50% loss of Rb occlusion, which correlates with partial reduction of S-S bridges in the extracellular portion of the beta-subunit; both of these effects were prevented by the presence of 20 mM RbCl. To clarify the role of the extracellular portion of the beta-subunit, "19-kDa membranes" (Na,K-ATPase posttryptic residues, which have been shown to possess many of the cation-binding properties) were used. Incubation of the "19-kDa membranes" with 0.2 M DTT for 1 h at 37 degrees C abolished 70-80% of the 86Rb occlusion capacity. This was accompanied by accumulation of 16- and 17-kDa peptides (in SDS-PAGE of the membranes) and release of a 45-kDa band derived from the Na,K-ATPase beta-subunit to the supernatant. The appearance of the 45-kDa fragment of the beta-subunit in the supernatant confirms the existence of only one transmembrane fragment in this subunit. N-Terminal sequence analysis of the 16- and 17-kDa bands revealed the same structure, A-K-E-E-G-, which corresponds to the beta-subunit sequence beginning at Ala5. The simultaneous presence of 25 mM RbCl (but not 25 mM choline chloride) during DTT treatment prevents almost all (85%) of the loss of Rb occlusion, the appearance of 16- and 17-kDa bands, and reduction and release of the 45-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:An essential role for the extracellular domain of the Na,K-ATPase beta-subunit in cation occlusion. 839 70

The bafilomycin A(1) and N-ethylmaleimide (NEM)-sensitive (V-type) ATPase was partially purified from the apical membrane-rich fractions of excretory system (Malpighian tubules and hind gut) of P. bufonius. Enzymatic activity was inhibited by bafilomycin A(1) (IC(50) = 1.3 nM) and NEM (IC(50) = 10.1 microM). The V-type ATPase activity is confined to the apical membrane fraction, while the activity of Na(+)/K(+) -ATPase forms the major part of the basal membrane fraction. The optimal pH required for maximal activity of V-type ATPase was pH 7.5. The effect of 30 mM of various salts on ATPase activity was investigated. NaCl and KCl caused increases of 175% and 184%, respectively. Other chloride salts also caused an increase in activity in the following ascending order: RbCl, LiCI, choline Cl, NaCI, KCl and tris-HCl. The activity of V-type ATPase was stimulated by a variety of different anions and cations, and HCO(3)(-) was found to be the most potent cationic activator of ATPase activity. The present results show that the properties of V-type ATPase of P. bufonius are similar to those reported for other insect tissues.
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PMID:Properties of the V-type ATPase from the excretory system of the usherhopper, Poekilocerus bufonius. 1221 49

We previously demonstrated that pinacidil does not affect Na(+)(i) accumulation, cellular energy depletion, or acidosis during myocardial ischemia, but dramatically improves the cationic/energetic status during reperfusion. We investigated the role of this latter effect in K(ATP) channel-induced cardioprotection. Employing (23)Na and (31)P nuclear magnetic resonance spectroscopy with perfused rat hearts, reperfusion Na(+)(i) was altered with brief infusions of ouabain and/or RbCl to transiently decrease or increase Na(+)/K(+) ATPase activity. The increases and decreases in functional recovery (%LVDP-R) with pinacidil or ouabain, respectively, were largely unaltered by each other's presence. Early reperfusion Na(+)(i) and cellular energy were greatly altered by ouabain and indicated linear relationships with %LVDP-R. Pinacidil shifted these relationships to higher %LVDP-R. Increasing early reperfusion Na(+)(i) decreased %LVDP-R but did not diminish pinacidil's capacity to improve %LVDP-R. Approximately 75% and 45% of the pinacidil-induced improvements in %LVDP-R, could be disassociated from early reperfusion Na(+)(i) and cellular energy, respectively. Both pinacidil and RbCl infusion blunted ouabain's elevation of reperfusion Na(+)(i), but RbCl did not improve %LVDP-R. Atomic absorption tissue Ca(2+) measurements indicated that pinacidil reduced late reperfusion Ca(2+) uptake, but did not reduce early reperfusion Ca(2+), and its beneficial effects were resistant to ouabain-induced early reperfusion Ca(2+) increases. In conclusion, K(ATP) channel-induced cardioprotection does not require moderation of Na(+)(i) accumulation, cellular energy depletion, or acidosis during ischemia. K(ATP) channel-induced cardioprotection is largely independent of the accelerated reperfusion Na(+)(i) recovery it induces and does not require early reperfusion reductions of tissue Ca(2+). A larger role for early reperfusion cellular energy cannot be excluded.
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PMID:Early reperfusion levels of Na(+) and Ca(2+) are strongly associated with postischemic functional recovery but are disassociated from K(ATP) channel-induced cardioprotection. 1527 18

A high-throughput screening (HTS) assay was developed for the Na(+),K(+)-ATPase channel in order to study rubidium uptake as a measure of the functional activity and modulation of this exchanger. The assay uses elemental rubidium as a tracer for K(+) ions. Three cell lines were used to study the exchanger, and the assay was performed in a 96-well microtiter plate format. Rb(+) uptake was carried by the CHO-K1 cells at 37 degrees C; the maximum ion influx was at 80 min of incubation of the cell line in the medium containing 5.4 mM RbCl. The cells were incubated in Rb(+) uptake buffer (5.4 mM) and with the pump blocker ouabain for 1, 2, and 3 h, respectively. A complete block of the Rb(+) uptake was observed with a 5 mM concentration of ouabain for all the three time intervals. The ouabain 50% inhibitory concentration (IC(50)) value for CHO-K1 cell line ATPase was observed to be 298 microM after 3 h of incubation. In addition, IC(50) values of 94 and 89 microM were observed at 30 min of incubation, indicating that the protocol shows reproducible results. A Z' factor higher than 0.7 was observed in the assays. These studies extend the profile of Na(+),K(+)-ATPases and demonstrate the feasibility of this HTS assay system to screen for compounds that pharmacologically modulate the function of Na(+),K(+)-ATPase.
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PMID:Development of an HTS assay for Na+, K+-ATPase using nonradioactive rubidium ion uptake. 1567 51

It was previously shown that the adrenergic agonist dobutamine stimulates Rb(+) uptake in isolated pig hearts. In the present work we examined whether dobutamine can increase Rb(+) uptake rate in vivo. Open-chest domestic pigs (N = 14) were used under general anesthesia. The surface coil was placed against the anterior left ventricular wall to obtain (87)Rb spectra using a spectrometer interfaced with the 7T, 30-cm horizontal bore magnet. RbCl was infused at the rate of 1.35 +/- 0.14 mmol/kg/hr without or with dobutamine (0.6 mg/kg/hr) over a 60-min period, and then the infusions were terminated. The rate constant (k x 10(3), from 13 +/- 2.4 to 36 +/- 12 min(-1)) and Rb(+) influx rate (from 2.5% to 4.8%/min) were increased by dobutamine despite lower plasma [Rb(+)] (0.59 +/- 0.16 vs. 0.84 +/- 0.24 mM in control), while the tissue/plasma Rb ratios were identical (38 +/- 9). Heart rate (HR) and systolic blood pressure (SBP) were increased from 106 +/- 9 to 161 +/- 15 bpm (+52%) and from 78 +/- 7 to 93 +/- 11 mmHg (+19%), respectively. The stimulation of Rb(+) uptake by dobutamine is consistent with the activation of Na(+)/K(+) ATPase previously observed in isolated hearts. However, the 50% increase in HR and the double coronary flow caused by the SBP increase and vasodilatation may also contribute to this effect.
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PMID:Adrenergic stimulation of Rb+ uptake in pig hearts in vivo assessed by 87Rb MRS. 1579 46


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