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)

Specific [3H]ouabain binding to rat and guinea pig skeletal muscle (musculus soleus) was studied using a rapid centrifugation and a filtration method. Both assays gave identical results: the incubation of the cell membranes in 50 mM imidazole/HCl buffer pH 7.25 or 7.4 MgCl2, Pi caused a time dependent loss of (Na+ +K+)-ATPase activity indicating an alteration of the membrane preparation. Ouabain binding properties were changed concomitantly. If ouabain binding was allowed to proceed until equilibrium was reached (3 min in rat and 10 min in guinea pig) at 37 degrees C the data plotted according to Scatchard followed a straight line. The dissociation constants of the ouabain-receptor-complexes of the rat cell membrane preparation as calculated from the slope of the plot (KD = 134 nM) and from the ratio of the dissociation and association rate constants (KD = 175 nM) agreed within experimental error with that determined by Clausen and Hansen [(1974) Biochim. Biophys. Acta 345, 387-404] in intact soleus muscles (KD = 210 nM). If ouabain binding was allowed to proceed for a longer period, however, nonlinear Scatchard plots resulted with an identical maximal number of binding sites but inconstant and decreased affinity for the cardiac glycoside. Experimental evidence is presented that nonlinear Scatchard plots often obtained in hormone (drug)-receptor binding experiments may (among other things) be the result of damaged cell membrane particles in vitro.
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PMID:Ouabain-receptor interactions in (Na+ + K+)-ATPase preparations. A contribution to the problem of nonlinear Scatchard plots. 13 90

Calcium binding at 0 degrees C to a purified sheep kidney Na+,K+-ATPase was described by linear Scatchard plots. Binding at saturating free calcium was 65-80 nmol/mg of protein, or 30-40 mol of calcium/mol of enzyme. Aqueous emulsions of lipids extracted from Na+,K+-ATPase yielded dissociation constants and maximum calcium-binding values that were similar to those for native Na+,K+-ATPase. Phospholipase A treatment markedly reduced calcium binding. Pretreatment of native Na+,K+-ATPase with ouabain increased the dissociation constant for calcium binding from 131 +/- 7 to 192 +/- 7 muM without altering maximum calcium binding. Ouabain pretreatment did not affect calcium binding to extracted phospholipids, ouabain-insensitive ATPases, or heat denatured Na+,K+-ATPase, Na+ and K+ (5-20 mM) increased the dissociation constants for calcium, which suggests competition between the monovalent cations and calcium for the binding sites. At higher concentrations of monovalent cations, ouabain increased the apparent affinity of binding sites for calcium. Extrapolation to physiological cation concentrations revealed that the ouabain-induced increase in apparent affinity for calcium may be as much as 2- to 3-fold. These results suggest: (1) calcium binds to phospholipids associated with Na+,K+-ATPase; (2) ouabain interaction with Na+,K+-ATPase induces a perturbation that is transmitted to adjacent phospholipids, altering their affinity for calcium; and (3) at physiological concentrations of Na+ or K+, or both, ouabain interaction with Na+,K+-ATPase may lead to an increased pool of membrane-bound calcium.
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PMID:A possible molecular mechanism of the action of digitalis: ouabain action on calcium binding to sites associated with a purified sodium-potassium-activated adenosine triphosphatase from kidney. 13 87

Previous studies on the urinary bladder of the seawater-acclimated winter flounder (pseudopleuronectes americanus) demonstrated that active Na and Cl transport were ouabain sensitive. This suggested a relationship between the Na pump and Na-K-ATP-ase. The specific binding of [H]ouabain to Na-K-ATPase provides a means of localizing the site of active Na transport. In isolated bladders, a positive linear correlation (r= 0.89) was found between the active Na transport rate and the Na-K-ATPase activity. Ouabain binding by the bladder surface appeared to be saturable and relatively specific, e.g., was reduced by a high K concentration. When only the mucosal side of the bladder was exposed to 5 muM ouabain, both inhibitory effects and binding were small and are explained by finite permeability of the bladder to ouabain. In contrast, binding and inhibitory effects from the serosal side were much greater. Autoradiographs demonstrated that [3H]ouabain was bound only to the serosal side of the epithelial cells. Ultrastructural examination revealed that the area of ouabain binding coincided with the basal and lateral plasma membranes.
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PMID:Na-K-ATPase localization in teleost urinary bladder by [3H]ouabain autoradiography. 13 77

1. Digitonin treated membrane preparations purified from dog kidney lose their (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, but the K+-phosphatase and Na+-dependent ADP-ATP exchange activities survive and remain ouabain-sensitive. Because the enzyme preparations consist largely of pure (Na+,K+)-ATPase, these effects of digitonin must be intrinsic to the Na+ pump. 2. Concomitant with these enzymatic changes, digitonin treatment alters the sensitivity of the phosphatase and exchange activities to ouabain. 3. Attempts to measure ouabain binding by the usual centrifugation or filtration methods proved unsuccessful. A filtration method involving a double 0.01 mum filter and omitting water washes is necessary to demonstrate ouabain binding. Under these conditions, ouabain binding capacity appears to be unchanged in the presence of digitonin, but the apparent dissociation constant is doubled. 4. Ouabain binding is rendered more reversible by digitonin treatment, since washing filters with water removes a large fraction of bound ouabain without affecting the retention of exchange activity. 5. The double filter method traps essentially all of the ADP-ATP exchange activity on the filter. However, a large and somewhat variable proportion of the K+-phosphatase activity passes through the filter. Sodium dodecyl sulfate polyacrylamide gel analysis of the filtrate shows that a small amount of filtrable protein catalyzed this phosphatase activity at greatly increased turnover rates. Both subunits of the (Na+, K+)-ATPase are present in this latter protein fraction.
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PMID:The measurement of ouabain binding and some related properties of digitonin-treated (Na+,K+)-ATPase. 13 44

Harmaline inhibits both the Na+ -K+ -ATPase activity and the uptake of L-phenylalanine in guinea-pig intestinal mucosa. The latter effect is not a direct consequence of the former, since higher concentrations are needed to inhibit the enzyme than the influx into the mucosa; Furthermore the uptake is still sensitive to harmaline when the Na+ -K+ -ATPase has been fully inhibited by ouabain. Harmaline can inhibit L-phenylalanine influx at a concentration at which it does not affect intracellular ion concentrations. Ouabain, however, inhibits the uptake of L-phenylalanine only after a 30 min preincubation period, when the intracellular sodium concentration reached the extracellular level. Harmaline also interferes with the influx of beta-methyl-D-glucoside in the mucosa of the dog colon. Addition of harmaline at the mucosal face of the tissue suppresses all net transport of sodium and chloride ions and L-phenylalanine across the mucosa. Thus the same mode of action appears to apply in both the guinea-pig ileum and the dog colon.
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PMID:Differential effects of harmaline and ouabain on intestinal sodium, phenylalanine and beta-methyl-glucoside transport. 13 95

The involvement of membrane (Na+ + K+)-ATPase (Mg2+-dependent, (Na+ + K+)-activated ATP phosphohydrolase, E.C. 3.6.1.3) in the oxygen consumption of rat brain cortical slices was studied in order to determine whether (Na+ + K+)-ATPase activity in intact cells can be estimated from oxygen consumption. The stimulation of brain slice respiration with K+ required the simultaneous presence of Na+. Ouabain, a specific inhibitor of (Na+ + K+)-ATPase, significantly inhibited the (Na+ + K+)-stimulation of respiration. These observations suggest that the (Na+ + K+)-stimulation of brain slice respiration is related to ADP production as a result of (Na+ + K+)-ATPase activity. However, ouabain also inhibited non-K+ -stimulated respiration. Additionally, ouabain markedly reduced the stimulation of respiration by 2,4-dinitrophenol in a high (Na+ + K+)-medium. Thus, ouabain depresses brain slice respiration by reducing the availability of ADP through (Na+ + K+)-ATPase inhibition and acts additionally by increasing the intracellular Na+ concentration. These studies indicate that the use of ouabain results in an over-estimation of the respiration related to (Na+ + K+)-ATPase activity. This fraction of the respiration can be estimated more precisely from the difference between slice respiration in high Na+ and K+ media and that in choline, K+ media. Studies were performed with two (Na+ + K+)-ATPase inhibitors to determine whether administration of these agents to intact rats would produce changes in brain respiration and (Na+ + K+)-ATPase activity. The intraperitoneal injection of digitoxin in rats caused an inhibition of brain (Na+ + K+)-ATPase and related respiration, but chlorpromazine failed to alter either (Na+ + K+)-ATPase activity or related respiration.
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PMID:Control of brain slice respiration by (Na+ + K+)-activated adenosine triphosphate and the effects of enzyme inhibitors. 13 40

The effects of thyroid hormone on guinea pig myocardial NaK-ATPase activity, transmembrane monovalent cation active transport, and cardiac glycoside binding were were examined. NaK-ATPase activities of left atrial and left ventricular homogenates of control and triiodothyronine (T3)-treated animals were determined, and compared to activities of skeletal muscle and liver. T3 administration was associated with a significant increase of 18% in left atrial and left ventricular NaK-ATPase specific activities. This increment was less than that noted in skeletal muscle (+42%) and liver (+30%). To determine if enhanced NaK-ATPase activity was accompanied by increased monovalent cation active transport, in vitro 86Rb+ uptake by left atrial strips and hemidiaphragms was measured. Transition from the euthyroid to the hyperthyroid state resulted in a 68% increase in active 86Rb+ uptake by left atrium, and a 62% increase in active uptake by diaphragm. Passive 86Rb+ uptake was not affected in either tissue. Ouabain binding by atrial and ventricular homogenates of T3-treated animals was increased by 19 and 17%, respectively, compared to controls, in close agreement with thyroid-induced increments in NaK-ATPase activiey. Taken together, these results are consistent with enhanced myocardial NaK-ATPase activity and monovalent cation activt transport due to an increase in the number of functional enzyme complexes.
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PMID:Thyroid-induced alterations in myocardial sodium-potassium-activated adenosine triphosphatase, monovalent cation active transport, and cardiac glycoside binding. 13 89

The oxygen requirement of the Na-K-ATPase-dependent sodium transport system was examined in anesthetized dogs infused with 15% mannitol-Ringer solutions at a rate of 35 ml/min. Because of renal vasodilatation and abolished autoregulation, filtered sodium (FNa) could be varied over a wide range by progressive aortic constriction. Sodium reabsorption (RNa) and renal oxygen consumption (RVO2) varied in proportion to FNa (r greater than 0.9). Ouabain, which inhibits Na-K-ATPases, reduced RVO2 by 45 +/- 6%. During subsequent aortic constriction, the ratio delta RNa/delta FNa averaged 0.45 (glomerulotubular balance) (r less than 0.9), whereas RVO2 was not significantly altered. Comparisons of deltaRNa/deltaFNa before and after ouabain administration, indicate that about half of an increase in sodium delivery to the distal nephron is reabsorbed by the Na-K-ATPase-dependent sodium transport system and that deltaRNa/deltaRVO2 (Na/O2 ratio) of this system averages 14.5 +/- 1.3. This Na/O2 ratio corresponds to 2.4 sodium ions transported per ATP dephosphorylated as found in other tissues.
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PMID:Oxygen requirement of renal Na-K-ATPase-dependent sodium reabsorption. 13 8

Using small, intact frog muscles, the basic properties of Na+ and K+ transport were shown to resemble those of the (Na+ + K+)Mg2+ATPase (EC 3.6.1.3) isolated from skeletal muscle. (a) External K+ is essential for Na+ exit and K+ entry after the muscles are Na+-loaded and K+-depleted; (b) the ouabain concentration causing maximum inhibition of recovery is the same for transport as for the inhibition of the isolated enzyme. Ouabain causes a decrease in the sorbitol space and causes muscle fibre swelling. Absence of Ca2+ and Mg2+ inhibits recovery of normal Na+ and K+ concentrations and increases the sorbitol space. Insulin stimulates K+ uptake and Na+ loss in intact muscles but has no effect on the isolated sarcolemmal (Na+ + K+)Mg2+ATPase. Absence of divalent cations, addition of external ATP and of insulin enhance the ouabain inhibition of recovery. Bound ouabain was measured using [3H]ouabain and [14C]sorbitol (to measure the extracellular space). The process of binding was slowly reversible and was saturable within a range of ouabain concentrations from 1.48 X 10(-7) to 5.96 X 10(-7) M. From the nonexchangeable ouabain bound, the density of glycoside receptors was estimated to be 650 molecules per square micrometre of membrane surface. The absence of divalent cations, addition of external ATP and of insulin significantly enhanced the amount of ouabain bound. Substitution of Na+ and K+ by choline greatly reduced the bound ouabain.
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PMID:Enhancement (by ATP, insulin, and lack of divalent cations) of ouabain inhibition of cation transport and ouabain binding in frog skeletal muscle; effect of insulin and ouabain on sarcolemmal (Na + K)MgATPase. 13 99

Ouabain circulating in blood inhibits Na-K-ATPase in the gills of seawater eels at a concentration similar to that necessary for inhibition in vitro. By contrast, a much higher concentration is required when ouabain is applied to the exterior of the gill. Inhibition by external ouabain occurs only when the drug gains access to the circulation of the fish, as evidenced by simultaneous inhibition of Na-K-ATPase in the kidney. These results suggest that the Na-K-ATPase of gill chloride cells faces inward, lining intracytoplasmic tubular channels continuous with the extracellular fluid. Inhibition of gill Na-K-ATPase by ouabain in intact salt water eels results in almost complete inhibition of the efflux of both Na+ and Cl-. The efflux is tritiated water was much less reduced, to 60% of normal. Since chloride is actively transported outward across the gill of seawater teleosts, it is suggested that active chloride transport is coupled to Na-K-ATPase. A neutral sodium chloride carrier is postulated that is energized by the movement of sodium from extracellular fluid down its electrochemical gradient into the chloride cell.
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PMID:Ouabain inhibition of gill Na-K-ATPase: relationship to active chloride transport. 13 54


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