Sodium and buffer cations inhibit dephosphorylation of (Na+ + K+)-ATPase

Effects of various cations on the dephosphorylation of (Na+ + K+)-ATPase, phosphorylated by ATP in 50 mM imidazole buffer (pH 7.0) at 22 degrees C without added Na+, have been studied. The dephosphorylation in imidazole buffer without added K+ is extremely sensitive to K+-activation (Km K+ = 1 microM), less sensitive to Mg2+-activation (Km Mg2+ = 0.1 mM) and Na+-activation (Km Na+ = 63 mM). Imidazole and Na+ effectively inhibit K+-activated dephosphorylation in linear competitive fashion (Ki imidazole 7.5 mM, Ki Na+ 4.6 mM). The Ki for Na+ is independent of the imidazole concentration, indicating different and non-interacting inhibitory sites for Na+ and imidazole. Imidazole inhibits Mg2+-activated dephosphorylation just as effective as K+-activated dephosphorylation, as judged from the Ki values for imidazole in the two processes. Tris buffer and choline chloride, like imidazole, inhibit dephosphorylation in the presence of residual K+ (less than 1 microM), but less effectively in terms of I50 values and extent of inhibition. Tris inhibits to the same extent as choline. This indicates different inhibitory sites for Tris or choline and for imidazole. These findings indicate that high steady-state phosphorylation levels in Na+-free imidazole buffer are due to the induction of a phosphorylating enzyme conformation and to the inhibition of (K+ + Mg2+)-stimulated dephosphorylation.