Effect of potassium on the conformational state of the complex of ouabain with sodium and potassium dependent adenosine triphosphatase

The effects of K ⁺ on the ouabain (Na ⁺ + K ⁺) ATPase [Mg ²⁺ 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 Mg ²⁺ 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 2 conditions approached each other at low temperatures, and consequently a K ⁺ effect not observed below 17° occurred. Phlorizin, which was 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 Mg ²⁺, were also stabilized by K ⁺, whereas those formed with Mg ²⁺ and P(i) were 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 Mg ²⁺ was reversible when K ⁺ was removed. Attempts to convert the ouabain enzyme complex prepared in the presence of Mg ²⁺ and P(i) 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 Mg ²⁺. 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 Mg ²⁺ and treated with KCl was dissimilar to the ouabain enzyme complex prepared with Mg ²⁺ and P(i).