The proteoliposomal steady state. Effect of size, capacitance and membrane permeability on cytochrome-oxidase-induced ion gradients

1. The flux pathways for H⁺ and K⁺ movements into and out of proteoliposomes incorporating cytochrome c oxidase have been investigated as a function of the electrical and geometrical properties of the vesicles. 2. The respiration-induced pH gradient (ΔpH) and membrane potential (Δψ) are mutually dependent and individually sensitive to the permeability properties of the membrane. A lowering or abolition of Δψ by the addition of valinomycin increased the steady-state level of ΔpH. Conversely, removal of ΔpH by the addition of nigericin resulted in a higher steady-state Δψ. 3. Vesicles prepared by sonication followed by centrifugation maintained similar pH gradients at steady state to those in vesicles prepared by dialysis, although the time taken to reach steady state was longer. Higher pH gradients can be induced in non-centrifuged sonicated preparations. 4. No significant differences were found in H⁺ and K⁺ permeability between proteoliposomes prepared by dialysis or by sonication. The permeability coefficient of the vesicle bilayers for H⁺ was 6.1 x 10^-4 cm · s^-1 and that for K⁺ was 7.5 x 10^-10 cm · s^-1. An initial fast change in internal pH was seen on the addition of external acid or alkali, followed by a slower, ionophore-sensitive, change. The initial fast phase can be increased by the lipid-soluble base dibucaine and the weak acid oleate. In the absence of ionophores, increasing concentrations of oleate increased the rate of H⁺ translocation to a level similar to that seen in the presence of nigericin. Internal alkalinization could also be induced by oleate upon the addition of potassium sulphate. 5. The initial, pre-steady-state and steady-state ΔpH and Δψ changes can be stimulated using a model in which the enzyme responds to both ΔpH and Δψ components of the protonmotive force. At steady state, the electrogenic entry of K⁺ is countered by electroneutral exit via a K⁺/H⁺ exchange. 6. The permeability coefficient, P(H), calculated from H⁺ flux under steady-state turnover conditions, was approx. 100 times higher than the corresponding 'passive' measurements of P(H). Under conditions of oxidase turnover, the vesicles appear to be intrinsically more permeable to protons.