Molecular Pharmacology, Vol 8, 18-29, Copyright © 1972 by the American Society for Pharmacology and Experimental Therapeutics

Inhibition of Sodium- and Potassium-Dependent Adenosine Triphosphatase by N-Ethylmaleimide

II. Effects on Sodium-Activated Transphosphorylation

¹ Departnment of Pharmacology, Faculty of Medicine, University of Toronto, Toronto 181, Ontario, Canada

An Na⁺-stimulated ADP-ATP exchange reaction could be demonstrated in the microsomes obtained from guinea pig kidney. In agreement with previous results, N-ethylmaleimide was found to affect the Na⁺-stimulated exchange reaction in two different ways, probably by reacting at two distinct sites on the (Na⁺ + K⁺)-dependent ATPase (EC 3.6.1.3). The reactivity of these two sites to N-ethylmaleimide depended on the conformational state of the transport enzyme system.

The ability of N-ethylmaleimide to distinguish between the inward-facing (E₁) and outward-facing (E₂) conformations of (Na⁺ + K⁺)-ATPase was used to determine the conformational state of the transport enzyme system in the presence of different physiological ligands, either individually or in combination. All physiological ligands except Mg⁺⁺ (i.e., Na⁺, K⁺, and ATP) stabilized the E₁ conformation of (Na⁺ + K⁺)-ATPase. The E₂ conformation of the enzyme could be obtained either by treatment with Mg⁺⁺ alone or by phosphorylation of (Na⁺ + K⁺)-ATPase to E₂-P.

ATP by itself did not appear to change the E₁ conformation of the enzyme to E₂ by occupancy of the "modifier" site, in either the presence or absence of p-nitrophenyl phosphate. Although both the E₂-P and E₂ forms of the enzyme have a high apparent affinity for ouabain, the glycoside binds to Na⁺-E₁-ATP and Mg⁺⁺-E₁-ATP or E₁-P to a significant extent.

The present findings support the hypothesis that Na⁺-dependent phosphorylation is part of the ATP-hydrolyzing activity of the (Na⁺ + K⁺)-ATPase and that in the presence of Na⁺ the E₂ conformation of the transport enzyme system may be obtained only by phosphorylation of the (Na₊ + K⁺)-ATPase.

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ACKNOWLEDGMENTS We are indebted to Professors H. Kalant and J. Manery Fisher for their helpful criticism in the preparation of the manuscript.