The effects of K+ on the ouabain-(Na+ + K+)-ATPase [Mg2+-dependent, (Na+ + K+)-activated ATP phosphohydrolase, EC 184.108.40.206] complex were studied using partially purified rat brain enzyme preparations. The dissociation rate of the [3H]ouabain-enzyme complex prepared with ATP, Na+, and Mg2+ decreased sharply with temperature between 37° and 22°in the absence of KCl. Potassium stabilized the complex at 37°. The dissociation rate of the [3H]ouabain-enzyme complex in the presence of KCl was temperature-insensitive. Thus the dissociation rates under these two conditions approached each other at low temperatures, and consequently a K+ effect not observed below 17°. Phlorizin, which has been shown to increase the K+ affinity of (Na+ + K+)-ATPase, enhanced the stabilizing effect of K+ on the ouabain-enzyme complex. Ouabain-enzyme complexes formed with substrates such as p-nitrophenyl phosphate, acetyl phosphate, or carbamyl phosphate, in the presence of Na+ and Mg2+, were also stabilized by K+, whereas those formed with Mg2+ and Pi weer not. The dissociation rates of [3H]ouabain from the enzyme in the presence of K+ were similar regardless of the phosphate ligand used to support the binding. The K+-induced stabilization of the ouabain-enzyme complex formed with ATP, Na+, and Mg2+ was reversible when K+ was removed. Attempts to convert the ouabain-enzyme complex prepared in the presence of Mg2+ and Pi from a K+-insensitive to a K+-sensitive conformation were unsuccessful. Deoxycholic acid partially antagonized K+ effects on the rates of [3H]ouabain binding and dissociation in the presence of ATP, Na+, and Mg2+. It is concluded that K+ stabilizes the ouabain-enzyme complex by altering its configuration and that this effect of K+ is closely related to its effect on the native phospho-enzyme. The stabilization appeared to result from a reduced accessibility of the ouabain binding site on the enzyme. The ouabain-enzyme complex prepared in the presence of ATP, Na+, and Mg2+ and treated with KCl was dissimilar to the ouabain-enzyme complex prepared with Mg2+ and Pi.
ACKNOWLEDGMENT The authors thank Mr. Roxy H.-M. So for expert technical assistance.
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