Proton Stoichiometry of Electron Transport in Rodent Tumor Mitoplasts¹

The mechanistic vectorial H⁺/O translocation ratios characteristic of energy-conserving sites 2 + 3 and site 3 of the respiratory chain of two tumor cell lines were determined using succinate and ferrocytochrome c, respectively, as electron donors. The measurements were carried out on mitoplasts in order to allow ferrocytochrome c free access to its binding site on the inner mitochondrial membrane. The tumor cell lines used were Ehrlich ascites tumor and the AS30-D ascites tumor. K⁺ was used as charge-compensating cation in the presence of valinomycin. The O₂ uptake rate measurements were made with a fast-responding membraneless electrode whose response time was closely matched with that of a pH electrode. The rates of O₂ uptake and H⁺ ejection during the apparent zero-order rate phase of respiration, analyzed by computer, were extrapolated to zero time. The observed H⁺/O ratios for succinate oxidation in both tumors exceeded 7 and approached 8 and the H⁺/O ratios for the cytochrome oxidase reaction closely approached 4.0, in agreement with data on normal mitochondria. However, the rates of H⁺ back decay in the tumor mitochondria are relatively high and may influence the net efficiency of oxidative phosphorylation under intracellular conditions.