Ouabain-inhibited ATPase

Figure 41-13. Stoichiometry of the Na+-K ATPase pump. This pump moves three Na ions from inside the cell to the outside and brings two K+ ions from the outside to the inside for every molecule of ATP hydrolyzed to ADP by the membrane-associated ATPase. Ouabain and other cardiac glycosides inhibit this pump by acting on the extracellular surface of the membrane. (Courtesy of R Post.)...

ATP -I- creatine = ADP + phosphocreatine (N-ethylglycocyamine can also act as acceptor mitochondrial enzymes, mechanism, overview [1] <5> enzyme is functionally coupled to ouabain-inhibited (Na, K )-ATPase [30] <5> kinetic model of reaction [33] <6> mechanism [55,76])... 

Saks, V.A. Lipina, N.V. Sharov, V.C. Smirnov, V.N. Chazov, E. Crosse, R. The localization of the MM isozyme of creatine phosphokinase on the surface membrane of myocardial cells and its functional coupling to ouabain-inhibited (Na, K )-ATPase. Biochim. Biophys. Acta, 465, 550-558 (1977)... 

The key experiment was one which would not be carried out until Skou learned of a paper published in German four years earlier, which showed that the movement of cations across the red cell membrane was inhibited by cardiac glycosides such as ouabain, plant alkaloids used for some 200 years in the therapy of heart failure. In 1957, Skou was unaware of this important finding, and wrote later that he had not done the crucial experiment to show Na/K ATPase as the transport system. When done, the experiment was decisive ouabain inhibited the ATPase activity exactly as it did the cation fluxes. This led to a flurry of activity in many biochemical laboratories and allowed Skou (nine years after his original publication) to write a review in which he concluded that the enzyme fulfilled the requirements for a system responsible for active transport of Na+ and K+ across the cell membrane. Thus the Na/K ATPase had the following properties ... 

A Zn2+-dependent, ouabain-inhibited ATPase was found in both rabbit kidney and calf brain preparations, thus confirming the observations of Atkinson and Lowe (6). A Co2+-dependent, ouabain-inhibited ATPase was present in the same preparations, thus confirming the observations of Rendi and Uhr (7). Also, a Ni2+-dependent, ouabain-inhibited ATPase was found. When assayed in the presence of the divalent cations, Co2+, Ni2+, and Zn2+, the Na-K ATPase activity in these relatively crude membrane preparations was explored, in some detail, and compared with the activity found with Mg2+ or Mn2+. 

The variation in Na-K ATPase activity with pH and with Na K ratio is shown in Figures 3 and 4. Although some differences are apparent in the curves obtained with the various divalent cations, the qualitative behavior is the same for all. However, the relative activating effect is pH dependent. The patterns of activity variation in Figures 3 and 4 could be duplicated using controls that contained choline acetate instead of Na+-K+ acetate and ouabain, and so they do not reflect variations in degree of ouabain inhibition. No activating effect under the conditions studied could be found for Ba2+, Ca2+, Cd2+, Cu2+, Fe2+, Pb2+, or Sr2+. 

Two inhibitors of the exchange mechanism and ATP hydrolysis are ouabain and vanadate. Ouabain binds only to that portion of ATPase that would be exposed to the extracellular surface in situ. It appears to stimulate the incorporation of orthophosphate into the same site that is phosphorylated by ATP under normal conditions, but ouabain inhibits all pump and hydrolytic... 

Digitoxin and ouabain are used to stimulate the heart muscle. They work by binding to the Na+/K+ ATPase and inhibiting its action. The result of this is that Na+ leaks back into the cell. When this happens, the cell tries to maintain the osmotic balance pumping the sodium out with the Na+ZCa pump. This pumps Ca + into the cell, which triggers muscle contraction. 

In order to assess the developmental increases in Na,K-ATPase activity and abundance, membrane fractions were prepared from fetal and neonatal animals as previously described (9). Na,K-ATPase activity was measured enzymatically as the ouabain inhibitable fraction of the umoles of inorganic phosphate liberated per mg protein per hour. Figure 1 summarizes the results obtained from three litters. Sodium pump activity increased 10 fold between 15.5 days gestation and 20 days of age, which is similar to that previously reported (6). Whether the increased activity is a consequence of an increased enzymatic turnover or increased sodium pump abundance cannot be concluded from this data. We next tested the hypothesis that the increase resulted from an increase in abundance of alpha and/or alpha+ forms of Na,K-ATPase. 

The effects of ouabain on a sodium and potassium dependent adenosine triphosphatase (Na,K-ATPase) were studied on heart enzymes isolated from a number of mammalian species. Ouabain inhibition in vitro was correlated with in vivo changes in heart and enzyme function at doses lower than those necessary to induce toxicity and arrhythmias. ... 

In the dog heart, cardiac glycosides also caused a dose dependent release of Ca " from intracellular membranes such as the mitochondria and sarcoplasmic reticulum. This release was proportional to the magnitude of the inotropic response. The increased intracellular Na/K ratio resulting from the ouabain inhibition of Na,K-ATPase may explain the positive inotropic effects of ouabain on the basis of Na+ stimulated release of membrane-bound intracellular Ca 56-S8 Nakamura and Schwartz observed in vitro increase of released Ca when the intracellular pH was increased. Increased Na" " or K concentrations did not alter Ca release. Intracellular acidosis due to anaerobic glycolysis during ischemia may decrease the release of calcium and reduce the contract bility of the heart muscle . 

In addition to VO4, the Na /K -ATPases are inhibited by ouabain (see), and the plasma membrane H -ATPases are iiihibited by dicyclohexyldiimide (DCCD) and diethylstilbestrol. 

The site of ouabain inhibition of the Na pump has been studied in some detail using site-specific and random-site mutagenesis. Ouabain binds to the outside surface of the pump in a partially K -competitive manner. It was well known that rat Na,K ATPase was relatively resistant to ouabain compared to other spedes. When various Na pumps were sequenced, it was found that the... 

FIGURE 10.8 A schematic diagram of the Na, K -ATPase in mammalian plasma membrane. ATP hydrolysis occurs on the cytoplasmic side of the membrane, Na ions are transported out of the cell, and ions are transported in. The transport stoichiometry is 3 Na out and 2 in per ATP hydrolyzed. The specific inhibitor ouabain (Figure 7.12) and other cardiac glycosides inhibit Na, K -ATPase by binding on the extracellular surface of the pump protein. 

ATPase inhibitor. In such patients, inhibition of the sodium pump in the cells lining the blood vessel wall results in accumulation of sodium and calcium in these cells and the narrowing of the vessels to create hypertension. An 8-year study aimed at the isolation and identification of the agent responsible for these effects by researchers at the University of Maryland Medical School and the Upjohn Laboratories in Michigan recently yielded a surprising result. Mass spectrometric analysis of compounds isolated from many hundreds of gallons of blood plasma has revealed that the hypertensive agent is ouabain itself or a closely related molecule ... 

The Na+/K+-ATPase is the only enzyme known to interact with CTS, which reversibly bind to the extracellular side of the Na+/K+-ATPase at the E2-P conformational state [E2-P ouabain] and inhibit ATP hydrolysis and ion transport (Fig. lb, step 4). 

Cells are normally kept at osmotic (water activity) equilibrium by the action of the Na-pump. Inhibition of the pump with the specific Na -K -ATPase inhibitor, ouabain, causes cell swelling as does inhibition of it by hypothermia. The intracellular environment contains a high concentration of K (100 to 120 mM, in most mammalian cells), lower concentrations of Na (about 10 to 30 mM), and high... 

Furthermore, it has been found that ouabain, convallatoxin, and cymarin inhibit the PTX-induced contraction of rabbit aortic vascular smooth muscle (79). These observations have also provided evidence for the involvement of Na ,K -ATPase on the contractile effect of PTX in smooth muscle. 

Figure 4.10 Effect of reduced glutathione (GSH) (0-1.0 mM) on Na/K ATPase inhibition associated with the addition of oxidized glutathione (GSSG) (1 me). Experiments were performed using an isolated bovine ventricular Na/K ATPase preparation. Na/K ATPase activity was quantified by the ouabain-sensitive hydrolysis of ATP to yield inorganic phosphate. The data are presented as means standard errors of the means (n = 6).

Peng, M., Huang, L., Xie, Z., Huang, W. H. and Askari, A. Partial inhibition of Na+/K+-ATPase by ouabain induces the Ca2+-dependent expressions of early-response genes in cardiac myocytes. /. Biol. Chem. 271 10372-10378,1996. 

The effects of Li+ upon hematopoiesis have been proposed to be due to two different systems modification of the activity of the membrane Na+/K+-ATPase, and the inhibition of adenylate cyclase. Monovalent cation flux, in particular Na+ transport, is known to influence the differentiation and proliferation of hematopoietic stem cells. For instance, ouabain, an effective inhibitor of the membrane Na+/K+-ATPase, blocks the proliferation of lymphocytes and has been shown to attenuate the Li+-induced proliferation of granulocyte precursors [208]. Conversely, Li+ can reverse the actions of amphotericin and monensin, which mediate Na+ transport and which inhibit CFU-GM, CFU-E, and CFU-MK colony formation in the absence of Li+ [209]. Therefore, the influence of Li+ upon normal physiological cation transport—for example, its influence upon Na+/K+-ATPase activity—may be partly responsible for the observed interference in hematopoiesis. 

Desaiah D, Gilliland IK, Ho IK, et al. 1980a. Inhibition of mouse brain synaptosomal ATPases and ouabain binding by chlordecone. Toxicol Lett 6 275-285. 

Besides by interfering with the general energy maintenance of the cell, impact of active transport during intracellular accumulation can also be investigated by direct inhibition of certain transport mechanisms. To detect contribution of Na+-dependent transport, the Na+/K+-ATPase, one of the primary active transport systems of the cell, can be inhibited by ouabain at 10 /uM. Higher amounts of the inhibitor may seriously impede with the viability of the cell. To obtain reliable results, the cells are pretreated with ouabain for 15 min prior to addition of the analyte. Usually, the inhibitory effect of ouabain lasts up to 45 min [29],... 

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