Amino Group Modification of (Na+ + K+)-ATPase

The effects of three amino group reagents on the activity of (Na+ + K+)-ATPase and its component K+-stimulated p-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In the absence of added ligands, or with either Na+ of Mg2+ present, the enzyme inactivation process follows complicated kinetics. In the presence of K+, Rb+, or Tl+, protection occurs due to a change of the kinetics of inactivation toward a first-order process. ATP protects against inactivation at a much lower concentration in the absence than in the presence of Mg2+ (P50 6 microM vs. 1.2 mM). Under certain conditions (100 microM reagent, 0.2 M triethanolamine buffer, pH 8.5) modification of only 2% of the amino groups is sufficient to obtain 50% inhibition of the ATPase activity. Modification of amino groups with ethylacetimidate causes a nonspecific type of inactivation of (Na+ + K+)-ATPase. Mg2+ and K+ have no effects, and ATP only a minor effect, on the degree of modification. The K+-stimulated p-nitrophenylphosphatase activity is less inhibited than the (Na+ + K+)-ATPase activity. Half-inhibition of the (Na+ + K+)-ATPase is obtained only after 25% modification of the amino groups. Modification of amino groups with acetic anhydride also causes nonspecific inactivation of (Na+ + K+)-ATPase. Mg2+ has no effect, and ATP has only a slight protecting effect. The K+-stimulated p-nitrophenylphosphatase activity is inhibited in parallel with the (Na+ + K+)-ATPase activity. Half-inactivation of the (Na+ + K+)-ATPase activity is obtained after 20% modification of the amino groups.