Molecular Pharmacology, Vol 10, 509-518, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

Effect of Potassium on the Conformational State of the Complex of Ouabain with Sodium- and Potassium-Dependent Adenosine Triphosphatase

¹ Department of Pharmacology, Michigan State University, East Lansing, Michigan 48824

The effects of K⁺ on the ouabain-(Na⁺ + K⁺)-ATPase [Mg²⁺-dependent, (Na⁺ + K⁺)-activated ATP phosphohydrolase, EC 3.6.1.3] 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 P_i 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 P_i from a K⁺-insensitive to a K⁺-sensitive conformation were unsuccessful. Deoxycholic acid partially antagonized K⁺ effects on the rates of [3^H]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 P_i.

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ACKNOWLEDGMENT The authors thank Mr. Roxy H.-M. So for expert technical assistance.

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