Association of Hydrogen Ion Ejection with Oxidative Phosphorylation by Mouse Liver Mitochondria

1. A method for the determination of photophosphorylation, which involves hydrogen ion change, has been applied to the phosphorylation associated with succinate oxidation by rotenone-treated mouse liver mitochondria. The determination of hydrogen ion uptake as a measure of adenosine diphosphate phosphorylation is considered valid since ( a ) the hydrogen ion uptake observed following ADP addition was completely inhibited by oligomycin, and ( b ) the values of ΔH + :ΔADP determined by adding a known amount of ADP and measuring pH change were in good agreement with the theoretical values obtained between pH 6.5 and 8.5. 2. The Sr ++ :ADP coefficient calculated from the quotient of Sr ++ :O and ADP:O was used to calculate an ADP:O corrected for energy leaks. A higher Sr ++ :O ratio was obtained in the presence of oligomycin, and, by application of the Sr ++ :ADP quotient to this ratio, a corrected ADP:O could be obtained, which is considered to represent better the potential for oxidative phosphorylation. 3. The stochiometry of proton ejection associated with Sr ++ accumulation was also determined, and in phosphate medium the value of ΔH + :ΔSr ++ was 0.67. In the absence of phosphate, the ratios were markedly dependent on the concentration of Sr ++ and mitochondria. The optimal ratios were observed at a low Sr ++ concentration under these conditions, and were approximately 1 with tris(hydroxymethyl)aminomethane chloride medium. With tris(hydroxymethyl)methylglycine buffer, the ratios were as great as 2. Acetate shifted the optimal ratio to an even lower Sr ++ concentration. These data were examined on the basis of current proposed models for the translocation of ions, and it was concluded that the intramitochondrial pH plays a primary role in the regulation of respiration and cation uptake.