Abstract
The hydrolysis of ATP by (Na+ + K+)-ATPase (EC 3.6.1.3) involves Na+-dependent phosphorylation of the microsomal protein, and its breakdown is accelerated by K+. N- Ethylmaleimide, a sulfhydryl reagent, inhibited (Na+ + K+)-ATPase by affecting either Na+-dependent phosphorylation or K+-sensitive dephosphorylation, depending upon the concentration of the inhibitor used and/or the presence of physiological ligands. In a ligand-free medium, lower concentrations of N-ethylmaleimide preferentially inhibited K+-sensitive dephosphorylation. To decrease Na+-dependent phosphorylation, a higher concentration of the inhibitor was required, but the rates of inhibition of ATP-hydrolyzing activity and K+-sensitive dephosphorylation were always similar.
Physiological ligands influenced the sensitivity of the inhibition of either phosphorylation or dephosphorylation to N-ethylamleimide. The rates of inhibition of ATP-hydrolyzing activity and dephosphorylation were increased by Na+ and decreased by K+. These effects of monovalent cations could be reversed by nucleoside di- and triphosphates. In the presence of Mg++ alone or with either inorganic phosphate, or of Na+ plus ATP, the ability of N-ethylmaleimide to inhibit phosphorylation was markedly increased, with a concomitant loss of its effect on the dephosphorylation step.
The results suggest that N-ethylmaleimide may differentiate between the two major conformational states of (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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