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)

Canine renal Na,K-ATPase was treated with ATP dialdehyde, "oxATP" (20 microM), as described by G. Ponzio, B. Rossi, and M. Lazdunski (1983, J. Biol. Chem. 258, 8201-8205). In this system, a by-product, formaldehyde, was the inactivator. We modified the system to minimize such inhibition and to speed up the reaction. oxATP itself inactivated the enzyme at a rate that was slow at first and later speeded up. We fitted a precursor-product model to the data. Labeling with [3H]oxATP indicated about three sites per alpha beta protomer at complete inactivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled enzyme showed radioactivity in many components, in the alpha and beta subunits and in small molecules at the tracker dye region. ATP (20 mM) prevented all labeling and inactivation. Ponzio et al. concluded that oxATP labels covalently an ATP binding site. Our experiments did not support this conclusion. Ouabain did not affect labeling. Sodium stimulated both inhibition and labeling more than potassium did, indicating a high-affinity ATP binding site, if any. But nucleotide specificity for preventing or producing inhibition did not correspond to nucleotide specificity for binding of ATP to the native enzyme. Blocking the ATP binding center with fluorescein isothiocyanate or fluorosulfonyl benzoyl adenosine had no effect on [3H]oxATP labeling. ATP also prevented [3H]oxATP labeling of bovine serum albumin or of integral-membrane proteins.
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PMID:Inhibition and labeling of sodium, potassium ATPase by the dialdehyde derivative of ATP. 253 59

Na+/K+ -ATPase, reconstituted into phospholipid vesicles, has been used to study the localisation of binding sites of ligands involved in the phosphorylation reaction. Inside-out oriented Na+/K+ -ATPase molecules are the only population in this system, which can be phosphorylated, as the rightside-out oriented as well as the non-incorporated enzyme molecules are inhibited by ouabain. In addition, the right-side-out oriented Na+/K+ -ATPase molecules have their ATP binding site intravesicularly and are thus not accessible to substrate added to the extravesicular medium. Functional binding sites for the following ligands have been demonstrated: (i) Potassium, acting at the extracellular side with high affinity (stimulating the dephosphorylation rate of the E2P conformation) and low affinity (inducing the non-phosphorylating E2K complex). (ii) Potassium, acting at the cytoplasmic side with both high and low affinity. The latter sites are also responsible for the formation of an E2K complex and complete with Na+ for its binding sites. (iii) Sodium at the cytoplasmic side responsible for stimulation of the phosphorylation reaction. (iv) Sodium (and amine buffers) at the extracellular side enhancing the phosphorylation level of Na+/K+ -ATPase where choline chloride has no effect. (v) Magnesium at the cytoplasmic side, stimulating the phosphorylation reaction and inhibiting it above optimal concentrations.
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PMID:Cation sidedness in the phosphorylation step of Na+/K+-ATPase. 254 45

The retinal pigment epithelium (RPE) interposed between the vascular system of the choroid and the neural retina performs a variety of functions essential for vision. In order to further elucidate the transport functions of the RPE, apical membranes were isolated from the RPE of the dogfish (Squalus acanthias) by differential precipitation with calcium. Na-K-ATPase, an apical marker enzyme in this tissue, was enriched 15-fold in the final membrane fraction. About 50% of the membranes form right-side-out vesicles in which the membrane has retained its in vivo orientation. Sodium uptake into these vesicles as determined by a rapid filtration method was stimulated 37% by the presence of a proton gradient across the membrane (pHi = 6.1, pHo = 8.1). The stimulation was also observed in membrane vesicles "short-circuited" with valinomycin and K. The pH gradient-dependent sodium uptake but not the uptake in the absence of a pH gradient was completely inhibited by 5 X 10(-4) M amiloride, and 56% inhibition was found at 10(-5) M amiloride. The uptake of 22Na was also strongly decreased in the presence of nonradioactively labelled sodium and lithium; potassium was without effect. pH gradient dependence, amiloride sensitivity, saturability and cation specificity of the sodium flux indicate the presence of a Na/H exchanger in the apical membrane of the retinal pigment epithelium. The presence of the Na/H exchange process might have important implications for the control of pH in the subretinal space, optimum intracellular pH of the RPE and the triggering of other functions of the RPE.
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PMID:A Na/H exchange mechanism in apical membrane vesicles of the retinal pigment epithelium. 255 38

Sodium-potassium adenosine triphosphatase (Na-K-ATPase) activity was measured by microassay, and the surface density of basolateral membranes was measured morphometrically in postglomerular segments of single tubules isolated from normally developing, intact mouse kidneys and from transfilter metanephric cultures. Proximal tubule Na-K-ATPase activity was 1092 +/- 480 pmol/mm per hour in newborn mice, increasing to 2462 +/- 258 in 1-week-old and 3470 +/- 578 pmol/mm per hour in adult mice. The Na-K-ATPase activity in newborn mice was approximately one-third of the activity in adult mice. Tubular Na-K-ATPase in transfilter metanephric culture was 972 +/- 536 pmol/mm per hour, a mean value almost identical to that in newborn mice. The surface density of basolateral cell membranes was 1.36 +/- 0.60 microns2/microns3 in newborn mice and 1.34 +/- 0.45 microns2/microns3 in 1-week-old mice, increasing to 2.70 +/- 0.98 microns2/microns3 in 4-week-old mice and 2.89 +/- 0.51 microns2/microns3 in adult mice. The surface density of tubular basolateral cell membranes in transfilter metanephric culture was 1.13 +/- 0.51 microns2/microns3, not significantly different from the surface density in newborn mice. The calculated mean surface area of basolateral membranes per unit tubular length was greater in cultures than in newborns, however, because total epithelial volume per unit length was significantly larger in the cultured tubules. Membrane surface area in intact mice increased with age, the surface area per unit length of tubule in adults being 4.6 times the area in newborn animals.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Renal tubular differentiation in mouse and mouse metanephric culture. II. Na-K-ATPase activity. 256 15

Sodium, potassium-activated adenosine triphosphatase (ATPase) activity and the sensitivity of rat myocardium to ouabain was studied in isoproterenol (IPR)-induced cardiac hypertrophy. IPR in a dose of 5 mg/kg was administered to rats intraperitoneally, once daily, for seven days. Left ventricular trabeculae originating from IPR-treated rats were significantly less sensitive than controls to ouabain-induced positive inotropy. In crude homogenate and sarcolemmal fractions the ATP hydrolysing activity both in the presence of Mg++ (basic) and Mg++, Na+, K+ (total) was significantly reduced in the heart of IPR-treated rats. The difference between the total and basic ATPase, i.e. the Na+, K(+)-stimulated portion of the activity was slightly reduced, but the sensitivity of Na+, K(+)-ATPase to ouabain remained unchanged. The results indicate that the well-known relation of sodium pump inhibition to positive inotropy in the heart of IPR-treated rats may not be valid.
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PMID:Changes in sarcolemmal adenosine triphosphatase activity and in ouabain sensitivity of rat myocardium in isoproterenol-induced cardiac hypertrophy. 256 64

The number and activity of erythrocyte ATPase-dependent sodium-potassium pump units were increased in obese subjects (p = 0.02). No link was observed between the number or activity of the pump units and hypertension. The ouabain-insensitive rubidium (i.e. potassium) transport was not associated with relative body weight or blood pressure status. Sodium-lithium countertransport correlated significantly with obesity but not with blood pressure status. In the hypertensive patients, before or after therapy with verapamil, hydrochlorothiazide, pindolol or atenolol there were no significant differences in cation transport. We propose that the correlation between obesity and essential hypertension cannot be explained by these two cation transport systems.
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PMID:Erythrocyte cation transport in obesity, hypertension, and during antihypertensive drug therapy. 257 70

This paper describes properties of a simple manual assay for Rb+ occlusion on renal (Na+ + K+)-ATPase. Rb+ occlusion is measured by applying the enzyme plus Rb+ (86Rb) mixture to a Dowex-50 cation exchange column at 0 degree C, and eluting the enzyme with occluded Rb+ using an ice-cold sucrose solution. The enzyme-Rb+ complex is quite stable at 0 degree C. This method is useful for measuring Rb+ occlusion under equilibrium binding conditions and slow rates of dissociation of the enzyme-Rb+ complex. The stoichiometry of Rb+ occluded per phosphorylation site is 2. Rb+ saturation curves are strictly hyperbolic, suggesting that the two Rb+ sites have very different affinities, one in the micromolar range and one in the tens of millimolar range. ATP shifts the Rb+ saturation curves to the right (control K0.5 100-200 microM; plus ATP, K0.5 0.8-1.4 mM, in a 100 mM Tris-HCl medium, pH 7.0) and reduces the maximal level occluded (control approx. 4 nmol/mg; plus ATP approx. 3 nmol/mg protein). Thus, as expected, ATP shifts the E(1)2Rb+-E2(2Rb+)occ equilibrium towards E1. Sodium ions at concentrations of up to 30 mM compete with the rubidium ions, KNa = 1.86 mM in the Tris-HCl medium. Na+ at higher concentrations (30-100 mM) has an added non-competitive antagonistic effect. At room temperature, Rb+ dissociates slowly from the enzyme, kobs = 0.08 s-1, in the presence of either Rb+ (20 mM) or Na, (100 mM). As expected, dissociation is greatly accelerated by ATP, the rate being to fast to be measured by this technique. (Na+ + K+)-ATPase proteolyzed selectively by chymotrypsin in a Na+ medium, occludes Rb+. For control and proteolyzed (Na+ + K+)-ATPase the Rb+ saturation curves are similar and the rates of dissociation of the enzyme-Rb+ complex are identical. The chymotryptic split appears to disrupt antagonistic interactions between cation and ATP binding domains, while the E1-E2 conformational transition of the unphosphorylated protein probably remains.
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PMID:Rb+ occlusion in renal (Na+ + K+)-ATPase characterized with a simple manual assay. 282 11

Sprague-Dawley rats were fed a basal AIN-76 diet containing 80, 200, 350, 500 or 650 mg of magnesium per kilogram of diet for 6 wk. Ventricular slices, as well as microsomal fractions, were prepared from the hearts and were used to determine sodium-potassium pump activity. Sodium-potassium pump activity was assessed in the microsomal membranes by determining the ouabain-inhibitable Na+, K+-ATPase activity and [3H]ouabain binding, and in the ventricular slices, by determining ouabain-sensitive 86Rb uptake under K+-free conditions. The ATPase activity increased with increasing dietary magnesium, so that in the hearts of those animals that were fed 500 and 650 mg of magnesium/kg diet, it was significantly greater than the activity in the hearts of the animals fed 80 and 200 mg/kg diet. Similarly, 86Rb uptake by heart slices from rats fed 500 and 650 mg of magnesium/kg diet was significantly greater than the uptake by heart slices from animals fed 80 and 200 mg/kg diet. [3H]Ouabain binding did not change with increasing dietary magnesium. Thus, magnesium deficiency appears to have no effect on the number of sodium-potassium pump sites, but does decrease the activity of the pump. It is suggested that this leads to an increase in intracellular Na+, resulting in a change in the membrane potential, and may contribute to the arrhythmias associated with magnesium deficiency.
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PMID:Effects of dietary magnesium on sodium-potassium pump action in the heart of rats. 282 28

This study investigates the effect of variations in mineralocorticoid as well as cell sodium delivery and uptake on Na-K-ATPase activity in the mouse medullary thick ascending limb of Henle (mTALH). Pharmacologic doses of the mineralocorticoid deoxycorticosterone acetate (DOCA) resulted in a 28% increase of Na-K-ATPase activity. Furosemide-induced inhibition of sodium uptake by the mTALH cell also resulted in Na-K-ATPase activity reduction (45%). Sodium deprivation did not cause a clear change in enzyme activity, either at 3 d or 2 wk, likely reflecting the result of the opposing influences of decreased sodium delivery and increased endogenous aldosterone. Finally, the behavior of Na-K-ATPase activity at 3 d of sodium deprivation in the mTALH contrasted with a 60% increase in activity observed in the cortical collecting tubule, a nephron segment known to be responsive to mineralocorticoid, and this heterogeneity of response may suggest an important role for the mTALH in maintaining salt homeostasis.
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PMID:Modulation of Na-K-ATPase activity in the mouse medullary thick ascending limb of Henle. Effects of mineralocorticoids and sodium. 283 Mar 16

1. We have previously shown that the hypertrophy of the kidney induced by a high protein diet consists of a preferential hypertrophy of the thick ascending limb (TAL) of Henle's loop. This might be related to an increase in the active salt transport by this segment. Sodium, potassium-dependent adenosine triphosphatase (Na+,K+-ATPase) activity was measured in TAL from kidneys of rats fed either a low (LP) or a high (HP) protein diet for several weeks. 2. Enzymatic activity was measured by microdensitometry, after appropriate cytochemical reaction, for an adenosine 5'-triphosphate (ATP) concentration of 0-66 mmol/l. Both activity per unit tubular length and mean activity per unit tissue volume were recorded. A calibration was designed to convert usual microdensitometry units (extinction) into conventional biochemical units (mol of product formed). 3. For non-limiting substrate concentrations, the Na+,K+-ATPase activity, expressed per unit length of tubule on the sections, was 50% higher in HP than in LP rats, an increase proportional to that of the simultaneously measured tubule diameter. When expressed per unit tubular volume, Na+,K+-ATPase activity was similar in both groups of rats. The dissociation constant for ATP was also similar in both groups. 4. Results show that a high protein diet induces an increase in Na+,K+-ATPase activity in TAL, thus enabling an enhanced NaCl transport in this segment. This increase in transport capacity is not due to an increase in the density of enzymatic units but to an increase in their number, in relation to the hypertrophy of the TAL.
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PMID:Effect of high protein intake on sodium, potassium-dependent adenosine triphosphatase activity in the thick ascending limb of Henle's loop in the rat. 283 Oct 8


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