Investigation of Na+,K+-ATPase on a solid supported membrane: the role of acylphosphatase on the ion transport mechanism

Charge translocation by Na+,K+-ATPase was investigated by adsorbing membrane fragments containing Na+,K+-ATPase from pig kidney on a solid supported membrane (SSM). Upon adsorption, the ion pumps were activated by performing ATP concentration jumps at the surface of the SSM, and the capacitive current transients generated by Na+,K+-ATPase were measured under potentiostatic conditions. To study the behavior of the ion pump under multiple turnover conditions, ATP concentration jump experiments were carried out in the presence of Na+ and K+ ions. Current transients induced by ATP concentration jumps were also recorded in the presence of the enzyme α-chymotrypsin. The effect of acylphosphatase (AcP), a cytosolic enzyme that may affect the functioning of Na+,K+-ATPase by hydrolyzing its acylphosphorylated intermediate, was investigated by performing ATP concentration jumps both in the presence and in the absence of AcP. In the presence of Na+ but not of K+, the addition of AcP causes the charge translocated as a consequence of ATP concentration jumps to decrease by about 50% over the pH range from 6 to 7, and to increase by about 20% at pH 8. Conversely, no appreciable effect of pH upon the translocated charge is observed in the absence of AcP. The above behavior suggests that protons are involved in the AcP-catalyzed dephosphorylation of the acylphosphorylated intermediate of Na+,K+-ATPase. [Copyright &y& Elsevier]