Tryptic digestion of the (Na + K)-ATPase is both sensitive to and modifies K+ interactions with the enzyme.

Tryptic digestion of the (Na + K)-ATPase in the presence of choline chloride or NaCl ("Na-type") and in the presence of KCl ("K-type") produced distinct patterns of peptide fragments and losses of catalytic activity. The K0.5 for K+ to shift digestion from the Na-type, and its sensitivity to dimethyl sulfoxide and Triton X-100, were consistent with K+ acting at sites on the cytoplasmic face of the enzyme through which the K-phosphatase reaction also is activated. Reagents favoring the E1 conformational states, oligomycin, Triton, and ATP, shifted the pattern toward the Na-type, whereas those favoring E2 states, dimethyl sulfoxide, MgCl2, and MnCl2, shifted the pattern toward the K-type. Na-type digestion caused a greater loss of K-phosphatase than (Na + K)-ATPase activity, and the residual K-phosphatase activity was more sensitive to inhibition by Triton and ATP but stimulated more by dimethyl sulfoxide and inhibited less by Pi and MnCl2; all these effects are consistent with such digestion shifting equilibria toward E1 enzyme states. Accordingly, the K0.5 for K+ to activate the (Na + K)-ATPase was increased. However, the K0.5 for the K-phosphatase was unchanged; this observation requires revision of previous formulations, and bears on additional aspects of enzyme activity as well.