Studies on (Na+ + K+)-activated ATPase. XLVI. Effect of cation-induced conformational changes on sulfhydryl group...

(1) (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.1.6.3) contains 34 sulfhydryl groups on the catalytic subunit, and two on the glycoprotein subunit. Under native conditions, only sulfhydryl groups on the catalytic subunit are accessible to modifying reagents. (2) The degree of inhibition of (Na+ + K+)-ATPase activity by N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic acid) depends on the cations present in the reaction medium. Mg2+ strongly enchances the inhibitory effects of both sulfhydryl reagents. The effects of Mg2+ on the inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) are counteracted by the addition of Na+ or K+. Na+ has no more effect than choline on the inhibition by 5,5'-dithiobis(2-nitrobenzoic acid), but it enhances the inhibitory effect of N-ethylmaleimide at low Na+ concentrations (less than 10 mM). Low concentrations of K+ (less than 10 mM) slightly protect the enzyme against modification. (3) Titration of residual sulfhydryl groups reveals that these ions do not only influence modification of essential sulfhydryl groups, but also that of sulfhydryl groups which are not essential for the enzyme activity. (4) These results indicate that Na+, K+ and Mg2+ have marked effects on the conformation of the catalytic subunit of (Na+ + K+)-ATPase. Various enzyme conformations can be induced, depending on the concentration and the kind of cation added. The largest effects are observed after addition of Mg2+.