Na+-Na+ exchange mediated by (Na+ + K+)-ATPase reconstituted into liposomes. Evaluation of pump stoichiometry and response to ATP and ADP

Abstract

(Na+ + K+)-ATPase from shark rectal glands reconstituted into lipid vesicles and oriented inside out catalyses an ouabain-sensitive Na+-Na+ exchange in the absence of intravesicular K+ when ATP is added extravesicularly. Intravesicular ouabain inhibited the exchange completely. This was also the case with digitoxigenin added to the vesicles. Intravesicular oligomycin inhibited the Na+-Na+ exchange partly in a fashion which was ATP dependent. The exchange is accompanied by a net hydrolysis of ATP with an apparent Km of 2.5 microM. ADP was found to give no stimulation of the Na+-Na+ exchange, contrarily, ADP inhibited the ATP-dependent exchange of Na+ both at optimal and supraoptimal ATP concentrations. When initial influx and efflux of 22Na was measured and the hydrolysis of ATP concomitantly determined a coupling ratio of 2.8:1.3:1 was found, i.e. 2.8 moles of Na+ were taken up (cellular efflux) and 1.3 moles of Na+ extruded (cellular influx) for each mole of ATP hydrolyzed. The electrogenic Na+-Na+ exchange generated a transmembrane potential which was measured with the fluorescent probe ANS (8-anilino-1-naphthalenesulfonic acid) to be 60 mV positive inside the liposomes (extracellular).