Abstract
Publisher Summary This chapter describes an approach that has been developed to determine the control that cytochrome c oxidase (COX) exerts on the rate of endogenous respiration in intact cells, as well as a method for analysis of oxidative phosphorylation in permeabilized cells. The experimental procedures described in the chapter are based on polarographic measurements of oxygen consumption. KCN titration of COX activity in intact cells can be applied to intact cells to study the control exerted by COX on endogenous respiration. In particular, the isolated COX activity is titrated with KCN after inhibiting the upstream segment of the respiratory chain with antimycin A and reducing the endogenous cytochrome c pool with tetramethyl-p-phenylene diamine, a membrane-permeant, one-equivalent electron donor, in the presence of an excess of ascorbate as the primary reducing agent. The titration curve thus determined is, then, compared to the KCN titration curve of the endogenous respiration, where the COX activity participates as a respiratory chain-integrated step.
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