Sensitivity of the (Na + + K +)-ATPase to state-dependent inhibitors : Effects of digitonin and triton X-100

Abstract

Treatment of a purified (Na + + K +)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na + + K +)-ATPase reaction inhibited more than the K +-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na + + K +)-ATPase reaction but not the K +-phosphatase reaction; however, treatment with digitonin made the K +-phosphatase reaction almost as sensitive to oligomycin as the (Na + + K +)-ATPase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K +-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K +-phosphatase reaction more than the (Na + + K +)-ATPase reaction). Both digitonin and Triton markedly increased the K 0.5 for K + as activator of the K +-phosphatase reaction, with little effect on the K 0.5 for K + as activator of the (Na + + K +)-ATPase reaction. In contrast, increasing the K 0.5 for K + in the K +-phosphatase reaction by treatment of the enzyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K +-phosphatase reaction by ATP and decreased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E 1 conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E 2 state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K 0.5 for K + in the K +-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na + plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na + plus nucleotide.