Datensatz

Zusammenfassung

We have used admittance analysis together with the black lipid membrane technique to analyze electrogenic reactions within the Na⁺ branch of the reaction cycle of the Na⁺/K⁺-ATPase. ATP release by flash photolysis of caged ATP induced changes in the admittance of the compound membrane system that are associated with partial reactions of the Na⁺/K⁺-ATPase. Frequency spectra and the Na⁺ dependence of the capacitive signal are consistent with an electrogenic or electroneutral E₁P ↔ E₂P conformational transition which is rate limiting for a faster electrogenic Na⁺ dissociation reaction. We determine the relaxation rate of the rate-limiting reaction and the equilibrium constants for both reactions at pH 6.2–8.5. The relaxation rate has a maximum value at pH 7.4 (∼320 s⁻¹), which drops to acidic (∼190 s⁻¹) and basic (∼110 s⁻¹) pH. The E₁P ↔ E₂P equilibrium is approximately at a midpoint position at pH 6.2 (equilibrium constant ≈ 0.8) but moves more to the E₁P side at basic pH 8.5 (equilibrium constant ≈ 0.4). The Na⁺ affinity at the extracellular binding site decreases from ∼900 mM at pH 6.2 to ∼200 mM at pH 8.5. The results suggest that during Na⁺ transport the free energy supplied by the hydrolysis of ATP is mainly used for the generation of a low-affinity extracellular Na⁺ discharge site. Ionic strength and lyotropic anions both decrease the relaxation rate. However, while ionic strength does not change the position of the conformational equilibrium E₁P ↔ E₂P, lyotropic anions shift it to E₁P.