Characterization of partially purified heart sarcolemmal Na+-K+-stimulated ATPase.
The Km value for the dog heart (Na+-K+)-ATPase was 0.31 mM (MgATP), whereas the values for the concentrations of K+ and Na+ varied from 1.2 to 2.7 mM and 12 to 20 mM for half-maximal activation, respectively. The concentrations of ouabain and calcium for 50 percent inhibition of (Na+-K+)-ATPase activity varied from 2.4 to 3.2 muM and 0.5 to 1.2 mM, respectively, the inhibitory effects of these agents were pH dependent. This preparation bound about 50 nmoles of 1-anilino-8-napthaline sulfonate (ANS)/mg of protein and exhibited fluorescence attributable to the ANS-enzyme complex. Cations such as Na+,K+,Ca++, and Mg++ increased ANS-enzyme fluorescence intensity and the number of ANS binding sites but decreased the apparent ANS binding constant. The enzyme activity, ANS binding, and ANS-enzyme fluorescence were decreased by phospholipase A, phospholipase C, and trypsin treatments. Although ouabain inhibited enzyme activity and ANS-enzyme fluorescence markedly, it caused only a slight depression in ANS binding. These results extend support for the allosteric nature of the cardiac (Na+-K+)-ATPase and provide evidence for conformational changes during its activation by Na+ and K+.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology*; D010042:Ouabain / Q000494:pharmacology; D010740:Phospholipases; D011188:Potassium / Q000494:pharmacology*; D011485:Protein Binding; D012508:Sarcolemma / Q000201:enzymology*; D012964:Sodium / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D010740:Phospholipases; D000251:Adenosine Triphosphatases; D008274:Magnesium; D011188:Potassium; D002118:Calcium