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.