Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase

Abstract

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg2+ plus K+. Mn2+ was less effective and Ca2+ could not substitute for Mg2+. However, adding Ca2+ with Mg2+ or substituting Mn2+ for Mg2+ activated it appreciably in the absence of added K+, and all three divalent cations decreased apparent affinity for K+. Inhibition by Na+ decreased with higher Mg2+ concentrations, when Ca2+ was added, and when Mn2+ was substituted for Mg2+. Dimethyl sulfoxide, which favors E2 conformations of the enzyme, increased apparent affinity for K+, whereas oligomycin, which favors E1 conformations, decreased it. These observations are interpretable in terms of activation through two cases of cation sites. (i) At divalent cation sites, Mg2+ and Mn2+, favoring (under these conditions) E2 conformations, are effective, whereas Ca2+, favoring E1, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K+, while Na+ at these sites favors E1 conformations. K+ increases the Km for substrate, but both Ca2+ and Mn2+ decrease it, perhaps by competing with K+. On the other hand, phosphatase activity in the presence of Na+ plus K+ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg2+ and Mn2+, but not by adding Ca2+; this is consistent with stimulation occurring through facilitation of an E1 to E2 transition, perhaps an E1-P to E2-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg2+ plus Na+ alone or Mg2+ plus low K+: this effect of oligomycin may reflect acceleration, in the absence of adequate K+, of an alternative E2-P to E1 pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.