The Influence of Respiration and ATP Hydrolysis on the Proton‐Electrochemical Gradient across the Inner Membrane of Rat‐Liver Mitochondria as Determined by Ion Distribution

1 A technique is described, based on the distribution of rubidium, acetate and methylammonium ions, for the simultaneous estimation of membrane potential and pH gradient across the inner membrane of mitochondria. The technique requires less than 0.5 mg mitochondrial protein and is independent of many factors which interfere with electrode determinations of protonmotive force (Δ p ). 2 With a limiting matrix volume of 0.4 μl/mg mitochondrial protein, the indicated value of Δ p for rat liver mitochondria is 228 mV in state 4, 170 mV in state 3, and –0.6 mV in the presence of rotenone and uncoupler. The relative contributions of the pH gradient and membrane potential are dependent on the availability of electrophoretically and electroneutrally translocatable species in the incubation medium. 3 In a sucrose‐based medium containing 0.5 mM KCl, rotenone and uncoupler, the technique indicated a membrane potential of + 85 mV and a pH gradient of + 1.46 (acidic in the matrix compartment). In state 4, under no conditions examined did the pH gradient contribute more than 50% of the total protonmotive force. 4 The hydrolysis of ATP generates an optimal Δ p of 220 mV. 5 The proton conductance of the inner membrane is potential dependent, increasing when Δ p is greater than 200 mV. 6 The extra‐mitochondrial phosphate potential sustainable by respiration was found to change in parallel to Δ p , but to exceed the latter parameter when based upon a stoichiometry of two protons translocated per ATP synthesised.