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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 vector characteristics of the interacting Na+, K+-ATPase and ouabaine were studied in experiments on the restored ghosts of erythrocytes. It is shown that the effect of K+ on the enzyme activity is the same as in cases of using ATP and p-nitrophenylphosphate (p-NPP) as phosphorylating agents. ADP removes the p-NPP induced inhibition with ouabain. This effect is explained rather by addition of ADP to the enzyme substrate centre than by a decrease in the concentration of E1 approximately P phosphoform. Incorporation of labelled orthophosphate into p-nitrophenol (NP) in the presence of Na+, K+-ATPase preparations was not detected. It is shown that antibodies against the fraction of the brain microsomes inhibit K+-NPPases to a much less extent than Na+, K+-ATPase. The digitonin treatment does not remove (Na++ATP)-dependent increase in the K+-NPPase activity. A conclusion is drawn that the mechanisms of p-NPP hydrolysis differs from the mechanism of ATP hydrolysis.
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PMID:[Interaction of p-nitrophenylphosphate with Na+,K+-ATPase]. 22 60

A procedure was developed for the isolation of cardiac sarcolemmal vesicles. These vesicles are enriched about ten-fold (with respect to the tissue homogenate) in K+-stimulated p-nitrophenylphosphatase, (Na+ + K+)-ATPase, 5'-nucleotidase activities and sialic acid content, all of which are believed to be components of the sarcolemma. The sarcolemma of tissue culture cardiac cells were radioiodinated and the distribution of this radioiodine paralleled the distribution of the other membrane markers above. There was very little contamination of the sarcolemmal fraction by sarcoplasmic reticulum (as judged by Ca2+-ATPase and glucose-6-phosphatase activities) or inner mitochondrial membranes (as judged by succinate dehydrogenase activity). There may, however, be some contamination by outer mitochondrial membranes (as judged by monoamine oxidase and rotenone-insensitive NADH cytochrome c reductase activities) which have rarely been monitored in cardiac sarcolemmal preparations. The purity of this preparation is good when compared with other cardiac sarcolemmal preparations. This preparation should be very useful in studying the roles of the cardiac sarcolemma (e.g. in excitation contraction coupling and Ca2+ binding).
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PMID:Isolation and characterization of cardiac sarcolemma. 22 23

The (Na+ + K+)-stimulated ATPase activity decreases with increasing pressure and a plot of the logarithm of the activity versus pressure shows a change in slope at a defined breakpoint pressure (Pb). The value of Pb increases linearly with increasing temperature. A dT/dP value of 27.7 +- 0.4 (S.D.) K/1000 atm is obtained. This is in very good agreement with the pressure shift for the melting transitions in phospholipids and aliphatic chains. This strongly indicates that an aliphatic chain melting process is involved in the breakpoint in the Arrhenius plot and pressure dependence of (Na+ + K+)-ATPase. The p-nitrophenyl phosphatase activity of this enzyme also decreases with pressure. In this case the plot of the logarithm of the activity versus pressure is linear without a break-point. The temperature dependence for (Na+ + K+)-ATPase was also studied in the presence of fluidizing drugs: desipramine and benzylalcohol. The presence of these drugs had no effect on the inflection point in the Arrhenius plot.
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PMID:Pressure effects on lipid-protein interactions in (NA+ + K+)-ATPase. 22 42

By means of a preparation technique based on the discontinuous sucrose density gradient, subcellular fractions were isolated from guinea pig intestinal smooth muscle cells. A fraction which distributed to a 33% sucrose layer showed relatively high activities of 5'-nucleotidase, Na+ . K+-ATPase and ouabain sensitive Na+ . K+-ATPase. The fraction had a low NaN3 sensitive Mg2+-ATPase activity. On the other hand, the high activity of glucose-6-phosphatase showed a broad distribution. Though the sucrose density gradient proceeded over a series of the fine layers, cross-contamination of microsome into the 33% sucrose fraction was not reduced. To reduce microsomal cross-contamination, another procedure was employed. The homogenization time of 77000 xg sediment to be layered on the top of the sucrose density gradients was prolonged. This procedure did not change the distribution of K+ activated p-nitrophenylphosphatase, K+ activated ouabain sensitive p-nitrophenylphosphatase and ouabain sensitive Na+ . K+-ATPase activities. The peak of NADH cytochrome c reductase activity was shifted to a 38% sucrose fraction from a 33% sucrose fraction and the activity of this marker enzyme in the 33% sucrose fraction decreased to 60% of that of the prior procedure.
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PMID:[Examination of plasma membrane-enriched fraction from guinea pig intestinal smooth muscle by means of some marker enzymes (author's transl)]. 23 74

The peripheral membrane protein fraction released by washing Acholeplasma laidlawii membranes with low-ionic strength buffers contained about 50% of the total membrane-bound ribonuclease and deoxyribonuclease activities. The ATPase, NADH oxidase and p-nitrophenylphosphatase activities remained bound to the membrane even when EDTA was added to the wash fluids, and thus appear to belong to the integral membrane protein group. Serving as a marker for peripheral membrane proteins, the membrane-bound ribonuclease activity was solubilized by bile salts much more effectively than the integral membrane-bound enzymes. On the other hand, the solubilized ribonuclease showed a much lower capacity to reaggregate with other solubilized membrane components to membranous structures. Yet, most of the ribonuclease molecules which were bound to the reaggregated membranes could not be released by low-ionic strength buffer. The reaggregated membranes differed from the native membranes in the absence of particles on their fracture faces obtained by freeze cleaving, and by their much higher labeling by the [125-I]lactoperoxidase iodination system. These results suggest that most of the proteins are exposed on the reaggregated membrane surfaces, with very little, if any, protein embedded in its lipid bilayer core. Enzyme disposition in the A. laidlawii membrane was studied by comparing the activity of isolated membranes with that of membranes of intact cells after treatment with pronase or with an antiserum to membranes. The data indicate the asymmetrical disposition of these activities, the ATPase and NADH oxidase being localized on the inner membrane surface, while the nucleases are exposed on the external membrane surface.
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PMID:Characterization of the mycoplasma membrane proteins. V. Release and localization of membrane-bound enzymes in Acholeplasma laidlawii. 23 52

K+ interactions with a rat brain (Na+ + K+)-dependent ATPase and the associated K+-dependent nitrophenyl phosphatase activity were examined. Classes of sites for K+ were distinguished, initially, on the basis of affinity estimated by kinetic analysis in terms of KO.5 (the concentration for half-maximal activation), and by K+-accelerated enzyme inactivation by F-minus, which permits evaluation of a dissociation constant for K+, KD. Moderate-affinity sites ("alpha sites"), with a KD near 1 mM, were demonstrable for the phosphatase activity and for the "free" enzyme. High-affinity sites ("beta sites"), with a KD near 0.1 mM, were seen for the overall ATPase activity and under conditions in which enzyme phosphorylation by substrate also occurs. Further differentiation between alpha and beta sites was made in terms of (i) the characteristic changes in affinity with pH, and (ii) the efficacy of Li+ relative to K+, Rb+, Cs+, and Tl+ at these two classes of sites. Low-affinity sites ("gamma sites") through which K+ inhibits enzymatic activity were also detectable, with a KD around 140 mM. These data are incorporated into a model for the reaction sequence to accommodate both transport processes and certain K+/ATP antagonisms.
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PMID:Functionally distinct classes of K+ sites on the (Na+ + K+)-dependent ATPase. 23 73

Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before secrose-density gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na+ + K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-alpha-oxoglutarate aminotransferase, L-asparte-alpha-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase.
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PMID:The isolation and partial characterization of the plasma membrane from Trypanosoma brucei. 48 94

The development of typical protrusions in isolated hepatocytes after incubation with phalloidin was prevented by phospholipase A (from bee venom). When cells were preincubated with low concentrations of phospholipase A and the enzyme was removed by washing, the number of cells affected by 10 microgram phalloidin/ml was markedly reduced. If the pretreated cells were allowed to recover after removal of phospholipase, the sensitivity to phalloidin returned to nearly normal values. Transient treatment of hepatocytes with sublytic concentrations of phospholipase A did not destroy cell membranes, whereas 5-fold higher concentrations of the enzyme produced large protrusions quite different from those appearing during phalloidin poisoning. These findings suggest that phosphatides are needed for the recognition of phalloidin by liver cells. A series of marker enzymes were analysed in isolated plasma membranes from rat liver after treatment with phospholipase A. Changes in the activities of K+ Na+-ATPase and of p-nitrophenylphosphatase were observed. Other membranal enzymes were not markedly influenced. The inhibitory effect of phospholipase A on the phalloidin response is discussed in context of earlier findings suggesting an evident role of a membranal protein for the recognition of phalloidin.
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PMID:Transient desensibilization of isolated hepatocytes against phalloidin by treatment with phospholipase A. 63 83

The effects of the naturally occurring flavonoid, quercetin, on gastric H+, K(+)-ATPase were investigated. Quercetin inhibited hog gastric H+, K(+)-ATPase and K(+)-stimulated p-nitrophenyl phosphatase (K(+)-pNPPase) activity in a dose dependent manner with IC50 values of 2.3 microM, and 6.0 microM respectively. The inhibition of H+, K(+)-ATPase by quercetin is competitive with ATP and is noncompetitive with K+. The steady-state phosphorylation level of the enzyme was also dose-dependently reduced by quercetin with an IC50 value of 4.5 microM. These results suggest that quercetin reduces the phosphorylated enzyme level by competition with ATP, and thereby inhibits the H+, K(+)-ATPase activity.
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PMID:Inhibition of gastric H+, K(+)-ATPase by quercetin. 128 50

We have used liposomes with incorporated pig kidney Na+,K(+)-ATPase to study vanadate sensitive K(+)-K+ exchange and net K+ uptake under conditions of acetyl- and p-nitrophenyl phosphatase activities. The experiments were performed at 20 degrees C. Cytoplasmic phosphate contamination was minimized with a phosphate trapping system based on glycogen, phosphorylase a and glucose-6-phosphate dehydrogenase. In the absence of Mg2+ (no phosphatase activity) 5-10 mM p-nitrophenyl phosphate slightly stimulated K(+)-K+ exchange whereas 5-10 mM acetyl phosphate did not. In the presence of 3 mM MgCl2 (high rate of phosphatase activity) acetyl phosphate did not affect K(+)-K+ exchange whereas p-nitrophenyl phosphate induced a greater stimulation than in the absence of Mg2+; a further addition of 1 mM ADP resulted in a 35-65% inhibition of phosphatase activity with an increase in K(+)-K+ exchange, which sometimes reached the levels seen with 5 mM phosphate and 1 mM ADP. The net K+ uptake in the presence of 3 mM MgCl2 was not affected by acetyl phosphate or p-nitrophenyl phosphate, whereas it was inhibited by 5 mM phosphate (with and without 1 mM ADP). The results of this work suggest that the phosphatase reaction is not by itself associated to K+ translocation. The ADP-dependent stimulation of K(+)-K+ exchange in the presence of phosphatase activity could be explained by the overlapping of one or more step/s of the reversible phosphorylation from phosphate with the phosphatase cycle.
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PMID:Phosphatase activity and potassium transport in liposomes with Na+,K(+)-ATPase incorporated. 130 62


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