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Proton Stoichiometry of Electron Transport in Rodent Tumor Mitoplasts¹

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

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.

¹ This work supported in part by a grant from USPHS National Institute of General Medical Sciences (GM05919) to B. R. and by Institutional Biomedical Research Grant RR 5378

² USPHS Fogarty International Fellow (1F05 TWO3573). Present address: Departmento de Bioquimica, Facultad de Medicina, Universidad de Chile, Santiago, Chile.

³ Fellow of the Consejo Nacional de Investigaciones Cientificas y Tecnicas de la Republica Argentina. Present address: Instituto De Investigaciones Cardiologicas, Universita de Buenos Aires, 1122 Buenos Aires, Argentina.

⁴ Deceased March 4, 1986.

Received 7/ 1/87. Revised 11/ 3/87. Accepted 11/ 4/87.