The effects of several ligands on the potassium-vanadate interaction in the inhibition of the ( [formula omitted] and the Na +, K + pump

Abstract

Inhibition by vanadate of the K +-dependent p- nitrophenylphosphatase activity catalyzed by the ( Na + + K +)- ATPase partially purified from pig kidney showed competitive behavior with the substrate. K + and Mg 2+ acted as cofactors in promoting that inhibition. Ligands which inhibited the K +-dependent p- nitrophenyl phosphate hydrolysis (Na +, nucleotide polyphosphates, inorganic phosphate) protected against inhibition by vanadate. The magnitude of that protection was proportional to the inhibition produced in the absence of vanadate. In the presence of only p- nitrophenyl phosphate and Mg 2+, or when the protective ligands were tested alone, the activation of p- nitrophenyl phosphate hydrolysis by K + followed a sigmoid curve in the presence as well in the absence of vanadate. However, the combination of 100 mM NaCl and 3 mM ATP resulted in a biphasic effect of K + on the p- nitrophenyl phosphate hydrolysis in the presence of vanadate. After an initial rise at low K + concentration, the p- nitrophenylphosphatase activity declined at high K + concentrations; this decline became more pronounced as the vanadate concentration was increased. This biphasic response was not seen when a nonphosphorylating ATP analog was combined with Na + (which favors the nucleotide binding) or with inorganic phosphate (a requirement for K + -K + exchange). Experiments with inside-out resealed vesicles from human red cells showed that in the absence of Na + plus ATP, K + promoted vanadate inhibition of p- nitrophenylphosphatase activity in a nonbiphasic manner, acting at cytoplasmic sites. On the other hand, in the presence of Na + plus ATP, the biphasic response of p- nitrophenyl phosphate hydrolysis is due to K + acting on extracellular sites. In vanadate-poisoned intact red blood cells, the biphasic response of the ouabain-sensitive Rb + influx as a function of the external Rb + concentration failed to develop when there was no Na + in the extracellular media. In addition, in the absence of extracellular Na +, external Rb + did not influence the magnitude of inhibition. The present findings indicate that external K + favors vanadate inhibition by displacing Na + from unspecified extracellular membrane sites.