Abstract
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.
ACKNOWLEDGMENTS We are indebted to Professors H. Kalant and J. Manery Fisher for their helpful criticism in the preparation of the manuscript.
- Copyright ©, 1972, by Academic Press, Inc.
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