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

AbstractCharge 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

Modifications induced by acylphosphatase in the functional properties of heart sarcolemma Na+,K+ pump

AbstractAcylphosphatase purified from cardiac muscle actively hydrolyzes the phosphoenzyme intermediate of heart sarcolemma Na+,K+-ATPase. This effect occurred with acylphosphatase amounts (up to 800 unitsmg membrane protein) that fall within the physiological range and the low value of the apparent Km (0.69 × 10−7 M) indicates a considerable affinity of the enzyme towards this specific substrate. Acylphosphatase addition to purified sarcolemmal vesicles significantly increased the rate of Na+,K+-dependent ATP hydrolysis. Maximal stimulation, observed with 800 unitsmg protein, resulted in an ATPase activity which was about 2-fold over basal value. The same acylphosphatase amounts significantly stimulated, in a similar and to an even greater extent, the rate of ATP driven Na+ transport into sarcolemmal vesicles. These findings lead to suppose that an accelerated hydrolysis of the phosphoenzyme may result in an enhanced activity of heart sarcolemmal Na+,K+ pump, therefore suggesting a potential role of acylphosphatase in the control of this active transport system