NA,K-ATPASE IN ARTIFICIAL LIPID VESICLES - COMPARISON OF NA,K AND NA-ONLY PUMPING MODE

Na,K-ATPase from rabbit kidney outer medulla was reconstituted in large unilamellar lipid vesicles by detergent dialysis. Vesicles prepared in the presence or absence of potassium allowed to study two different transport modes: the (physiological) Na,K-mode in buffers containing Na+ and K+ and the Na-only mode in buffers containing Na+ but no K+. The ATP hydrolysis activity was obtained by determination of the liberated inorganic phosphate, Pi, an the inward directed Na+ flux was measured by 22Na-tracer flux. Electrogenic transport properties were studied using the membrane potential sensitive fluorescence-dye oxonol VI. The ratio v(Na,K) v(Na of the turnover rates in the Na,K-mode and in the Na-only mode is 6.6 ± 2.0 under otherwise identical conditions and nonlimiting Na+ concentrations. Strong evidence is found that the Na-only mode exhibits a stoichiometry of 3Nacyt +/2Naext +/1ATP, i.e. the exrtracellular (= intravesicular) Na+ has a potassium-like effect. In the Na-only mode one high-affinity binding side for ATP (KM ≅ 50 nM) was found, in the Na,K-mode a high- a low-affinity binding side with equilibrium dissociation constants, KM, of 60 nM and 13 μM, respectively. The sensitivity against the noncompetitively inhibiting ADP (K1 = 6 μM) is higher by a factor of 20 in the Na-only mode compared to the Na,K-mode. From the temperature dependence of the pumping activity in both transport modes, activation energies of 160 kJ/mol for the Na,K-mode and 110 kJ/mol for the Na-only mode were determined. © 1990.