AUTOREGULATION OF THE PHOSPHOINTERMEDIATE OF NA+/K+-ATPASE BY THE AMINO-TERMINAL DOMAIN OF THE ALPHA-SUBUNIT

Chymotryptic cleavage of the α-subunit of the canine kidney Na+/K+-ATPase in the presence of Na+ abolishes ATPase activity and yields an 83 kDa peptide from Ala 267 to the COOH-terminus. To test the proposal that E1 to E2 conformational transition is blocked in this modified enzyme, we have made a detailed comparison of its phosphorylation with that of the native enzyme by ATP. While phosphorylation of α is dependent on Na+ and prevented by K+, that of the 83 kDa peptide is modestly stimulated by Na+; and only this stimulation, but not the Na+-independent phosphorylation is inhibited by K+. Ouabain, which inhibits α-phosphorylation by ATP, activates Na+-independent phosphorylation of the 83 kDa peptide by ATP, and inhibits the Na+-stimulation of this process. While there is a ouabain-stimulated phosphorylation of α by Pi, the 83 kDa peptide is not phosphorylated by Pi with or without ouabain. In its sensitivity to ADP, and insensitivity to K+, the phosphopeptide is similar to the E1P of the native enzyme; however, the spontaneous decomposition rate of the phosphopeptide is orders of magnitude lower than that of the native EP. Na+ has no effect on the spontaneous decomposition of the phosphopeptide; but at high Na+ concentrations (K0.5 = 350 mM) the ADP sensitivity of the phosphopeptide is reduced. The phosphopeptide, like the native EP, is acid-stable, alkaline-labile, and sensitive to hydroxylamine and molybdate. The chymotrypsin-treated enzyme catalyzes an ADP-ATP exchange activity that is stimulated by Na+. The Na+-independent part of this exchange, unlike that of the native enzyme, is activated by ouabain. Our findings establish that (a) the phosphorylation process and its control by Na+, K+ and ouabain are autoregulated by the NH2-terminal domain of the α-subunit; and (b) the often repeated assumption that the primary role of this domain is in the regulation of E1-E2 transitions is not valid. © 1990.