Item

Abstract

(H,K)-ATPase containing membranes from hog stomach were attached to black lipid membranes. Currents induced by an ATP concentration jump were recorded and analyzed. A sum of three exponentials (τ^-1₁ ≈ 400 sec⁻¹, τ ^-1₂ ≈ 100 sec⁻¹, τ ^-1₃ ≈ 10 sec⁻¹; T = 300 K, pH 6, MgCl₂ 3 mm, no K⁺) was fitted to the transient signal. The dependence of the resulting time constants and the peak current on electrolyte composition, ATP conversion rate, temperature, and membrane conductivity was recorded. The results are consistent with a reaction scheme similar to that proposed by Albers and Post for the NaK-ATPase. Based on this model the following assignments were made: τ₂ corresponds to ATP binding and exchange with caged ATP. τ ₁ describes the phosphorylation reaction E₁ · ATP → E₁P. The third, slowest time constant τ ₃ is tentatively assigned to the E₁P → E₂P transition. This is the first electrogenic step and is accelerated at high pH and by ATP via a low affinity binding site. The second electrogenic step is the transition from E₂K to E₁H. The E₂K ↔ E₁H equilibrium is influenced by potassium with an apparent K _0.5 of 3 mm and by the pH. Low pH and low potassium concentration stabilize the E₁ conformation.