Relationship of transmembrane pH and electrical gradients with respiration and adenosine 5'-triphosphate synthesis in mitochondria

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Abstract
The mechanism of mitochondrial oxidative phosphorylation and its regulation were studied using suspensions of isolated rat liver mitochondria. Parallel measurements were made of mitochondrial volume, respiration, transmembrane pH and electrical gradients and ATP, ADP and Pi concentrations under various experimental conditions. The transmembrane electrical gradients were calculated from the equilibrium distributions of [3H]-triphenylmethylphosphonium (TPMP+), [3H]tribenzylmethylammonium (TBMA+) and K+ (plus valinomycin). The transmembrane distributions of labeled acetate, methylamine and 5,5-dimethyloxazolidine-2,4-dione were used for the calculation of pH gradients. The respiratory rate is strictly correlated with [ATP] ([ADP][Pi]) (free energy of ATP synthesis); there is no consistent correlation between the transmembrane electrical potential, the pH gradient or the total protonmotive force and the respiratory rate. Thermodynamic analysis indicates that, for the proton electrochemical gradient to serve as an intermediate in ATP synthesis, from 3-7 H+ would have to be transported per each ATP synthesized, depending on the experimental conditions. Apparently, the proton electrochemical gradient may not serve as a primary intermediate in oxidative phosphorylation.