Molecular Pharmacology, Vol 8, 149-158, Copyright © 1972 by the American Society for Pharmacology and Experimental Therapeutics

Evidence for Two Forms of Fluoride-Treated Sodium- and Potassium-Dependent Adenosine Triphosphatase

¹ Laboratory of Chemical Pharmacology, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014

The (Na⁺ + K⁺)-dependent ATPase (EC 3.6.1.3) from rat kidney microsomal fraction was inactivated by 5 mM Mg⁺⁺ and 10 mM F^- after incubation for about 1 hr at pH 8.5, but lower pH values or the presence of 30 mM KCl enhanced the enzyme activity. The inhibited enzyme was not appreciably reactivated by repeated washing of the particles with 0.1 M Tris buffer, pH 7.4, at 0°, or by incubation with buffer at 37°.

Regeneration rates measured in the presence of Na⁺ rose with increasing concentrations of this cation and were greatly enhanced by the inclusion of 15 mM Tris-ATP in the incubation medium. Preparations which had been inactivated by F^- in the presence of 30 mM K⁺ or had been temporarily exposed to K⁺ after prior inactivation recovered activity at about twice the rate of inhibited particles that had never been exposed to K⁺. Thus, after 10 min at 37° in 30 mM Na⁺ and 15 mM Tris-ATP, particles originally inhibited in the presence of 30 mM K⁺ regained 54% of the activity of controls, and preparations inhibited by NaF alone regained 22%. When 30 mM K⁺ was added to the solution of 30 mM Na⁺ and 15 mM ATP in which regeneration was attempted, no reactivation of the enzyme occurred.

The data suggest that K⁺ converts the inactivated enzyme to a K⁺ complex that resists reactivation, and that this reaction is associated with a change in conformation of the enzyme. After this change in conformation, the enzyme-potassium complex would appear to have a dissociation constant of the order of 2 x 10^-4 M. The affinity for potassium is thus sufficiently low so that washing of the particulate enzyme would be expected to remove this cation. The enzyme is then left in a form that regenerates rapidly with Na⁺ and ATP.