Re-evaluation of the H+/site ratio of mitochondrial electron transport with the oxygen pulse technique.

摘要: The number of protons ejected per pair electrons passing each energy-conserving site in the electron transport chain (the H+/site ratio) has been investigated rat liver mitochondria by means oxygen pulse technique introduced Mitchell and Moyle (1967) (Biochem. J. 105, 1147-1162). usual values 2.0 observed this method were found to be substantially underestimated as a result influx phosphate into mitochondria. This was shown three different kinds experiments. 1. Addition N-ethylmaleimide or mersalyl, inhibitors mitochondrial transport, increased ratio from 3.0. dependence effect on concentration either inhibitor identical with that for inhibition transport. Added diminished below absence N-ethylmaleimide. N-Ethylmaleimide protected elevated 3.0 against deleterious added phosphate, but did not prevent lowering weak acid anions such 3-hydroxybutyrate. 2. Prior washing remove endogenous leaks out during anaerobic preincubation led ratios near 3.0, which low concentrations phosphate-depleted decreased 2.0; addition returned 3. Lowering temperature 5 degrees, slows down even movements dependent any narrowly limited set experimental conditions. It occurred Ca2+ K+ (in presence valinomycin) mobile permeant cation. independent succinate, oxygen, mitochondria, rotenone, additions Ca2+, Li+, Na+ medium pH between 6.5 7.5. Inhibitors ions acids other than affect ratio. These results indicate re-uptake lost preincubation, reduces H+ ejection leads method. When are eliminated procedures described above, is about value appears closer true primary process