1. (Na,K)ATPase from kidney membranes has been reconstituted into proteoliposomes following solubilization in cholate, by the freeze—thaw sonication procedure described by Kasahara & Hinkle (1977). The method is rapid and convenient.2. Upon addition of ATP to the exterior medium the reconstituted vesicles sustain high rates of active ²²Na uptake and ⁸⁶Rb efflux with many properties similar to those of the Na/K pump in well characterized cells such as erythrocytes.3. Observations on both active and passive transport of ²² Na and ⁸⁶Rb indicate that the vesicle population is heterogeneous; about 40 per cent contain Na/K pumps and the remainder seem to be plain lipid vesicles.4. The major Na⁺‐ or K⁺‐stabilized non‐phosphorylated conformational forms of the (Na, K)ATPase, E₁. Na and E₂. (K) respectively, have been investigated in the proteoliposomes, with particular regard to sidedness of the actions of Na⁺ and K⁺.5. Tryptic digestion of the vesicles reveals the Na⁺‐ and K⁺‐stabilized conformations E₁. Na and E₂. (K) as characterized originally for purified (Na, K)ATPase (Jørgensen, 1975). Controlled trypsinolysis of Tris⁺‐loaded vesicles in a Na⁺‐or K⁺‐containing medium leads to typical biphasic (Na⁺) or simple exponential (K⁺) time courses respectively, for loss of ATP‐dependent ²²Na uptake (assayed subsequent to the tryptic digestion in the presence of inophores valinomycin plus FCCP). Tryptic digestion of K⁺‐ or Rb⁺‐ or Tris⁺‐loaded vesicles suspended in a Na⁺ medium results only in the biphasic (E₁. Na) pattern of loss of ATP‐dependent ²²Na uptake.6. ATP‐dependent ²²Na uptake and ⁸⁶Rb efflux are reduced by about the same extent following a short tryptic digestion in a Na⁺‐containing medium.7. Vanadate ions inhibit ATP‐dependent ²²Na uptake into the vesicles, at low concentrations (K_0·5 ∼ 2 × 10⁻⁷ m), following pre‐incubation together with Mg²⁺ and K⁺ ions. K⁺ ions in the medium are effective, K⁺ ions within the vesicle are not. Na⁺ ions in the medium prevent inhibition by vanadate+Mg²⁺ but do not reverse inhibition in vesicles pre‐incubated with vanadate, Mg²⁺ and K⁺ ions.8. The results show that the conformational forms E₁. Na and E₂. (K) are stabilized by Na⁺ or K⁺ ions respectively, bound to sites on the Na/K pump normally facing the cytoplasm. The significance of this finding is discussed in relation to the cation transport function of the pump.

Publications with shared reference:

0 publications found