H+ stoichiometry of sites 1 + 2 of the respiratory chain of normal and tumor mitochondria.
The mechanistic stoichiometry for vectorial H+ ejection coupled to electron transport through energy-conserving segments 1 + 2 was determined on cyanide-inhibited mitochondria from rat liver, rat heart, and Ehrlich ascites tumor cells, and on rat liver mitoplasts with ferricyanide or ferricytochrome c as electron acceptors. K+ (+ valinomycin) and Ca2+ were employed as permeant cations. Three different methods were employed. In the first, known pulses of ferricyanide were added, and the total H+ ejected was determined with a glass electrode. Such measurements gave H+/2e-values exceeding 7.0 for both normal and tumor mitochondria with beta-hydroxybutyrate and other NAD-linked substrates; uptake of Ca2+ was also measured and gave the expected q+/2e-ratios. The second type of measurement was initiated by addition of ferricytochrome c to rat liver mitoplasts, with H+ ejection monitored with the glass electrode and ferricytochrome c reduction by dual-wavelength spectrophotometry; the H+/2e-ratios generally exceeded 7.0. In the third type of measurement, mixing and dilution artifacts were eliminated by oxidizing ferrocytochrome c in situ with a small amount of ferricyanide. H+/2e-ratios for rat liver mitoplasts oxidizing beta-hydroxybutyrate consistently approached or exceeded 7.5. Over 150 measurements made under a variety of conditions gave observed H+/2e-ejection ratios significantly exceeding 7.0, which correlated closely with H+/2e-measurements on sites 1 + 2 + 3, sites 2 + 3, and site 2. Factors leading to the deficit of the observed ratios from the integral value 8 for sites 1 + 2 were discussed.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism* / Q000648:ultrastructure; D002412:Cations; D003574:Cytochrome c Group / Q000378:metabolism; D004579:Electron Transport; D005292:Ferricyanides / Q000378:metabolism; D066298:In Vitro Techniques; D008929:Mitochondria, Heart / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism*; D011522:Protons; D051381:Rats
D002412:Cations; D003574:Cytochrome c Group; D005292:Ferricyanides; D011522:Protons; D002118:Calcium