Two classes of ouabain binding sites in ferret heart and two forms of Na+-K+-ATPase.
In partially purified Na+-K+-adenosinetriphosphatase (ATPase) obtained from ferret heart, ouabain produced a monophasic inhibition curve; however, the curve spanned over 5 logarithmic units, indicating the presence of more than one classes of enzyme. [3H]ouabain binding studies revealed high-and low-affinity binding sites in approximately equal abundance, with apparent dissociation constants of 10 and 230 nM, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of phosphoenzyme formed from [gamma-32P]ATP showed two distinct K+-sensitive bands of approximately 100,000 molecular weight. Phosphoenzyme formation from the high-molecular-weight alpha(+) form was selectively inhibited by N-ethylmaleimide. Ouabain caused a 50% inhibition of phosphorylation of the alpha(+) form at 40 nM and the lower-molecular-weight alpha form at 300 nM. In papillary muscle preparations, 1-30 nM ouabain produced a modest positive inotropic effect that reached an apparent plateau at 30 nM. Further increases in ouabain concentrations, however, produced additional and prominent inotropic effects at 0.1-10 microM. These results indicate for the first time in cardiac muscle that the high- and low-affinity ouabain binding sites are associated with the alpha(+) and alpha forms of the Na+-K+-ATPase, respectively, and that binding of ouabain to either of these sites causes enzyme inhibition and the positive inotropic effect.
D000818:Animals; D001665:Binding Sites; D005033:Ethylmaleimide / Q000494:pharmacology; D005289:Ferrets; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D008968:Molecular Conformation; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D010042:Ouabain / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014316:Tritium
D007527:Isoenzymes; D014316:Tritium; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D005033:Ethylmaleimide