Reconstitution of the Na+, K+-transport system in artificial membranes

(1) Purified renal Na+, K+-ATPase was incorporated into phosphatidylcholine-liposomes by using the cholate-dialysis procedure. The passive permeability of the liposomes for Na+ and K+ was determined on 22Na and 86Rb equilibration curves. The rate constant for passive Na-flux was 0.014 min-1 and for K(86Rb)-flux 0.013 min-1. Active transport rates of Na+ and K+ were determined after addition of 3 mM ATP to the liposomes. (2) A micromethod for continuously measuring the ADP production associated with active transport is described. Using this technique, initial velocities of ATP hydrolysis were measured in parallel with Na+-transport and K+-transport. When the Na+-concentration in the incubation medium is increased, the initial velocity of all three processes increase concomitantly. At high turnover, the stoichiometry of coupled Na+, K+-transport catalyzed by the purified reconstituted pump is 2.2Na+: 1K+:1ATP. (3) When the pump has exhausted the vesicular K+-pool, coupled NA+, K+-transport ceases. The pump then carries Na+ uphill without K+-antiport. The Na+: ATP ratio of uncoupled Na+-transport is close to 1:1.