Isolation of an Oligomycin-Sensitive ATPase Complex from Rat Liver Mitochondria

Although the antibiotic oligomycin blocks the terminal step in oxidative phosphorylation and inhibits ATPase activity of submitochondrial particles, purified F₁ ATPase preparations isolated from rat liver mitochondria are insensitive to oligomycin. Because the adenosine triphosphate (ATP) synthetic and hydrolytic activities of this enzyme in situ are affected by oligomycin, a more complete understanding of its physiological role requires that the isolated enzyme retain inhibitor sensitivities similar to those seen in mitochondria. This chapter describes a procedure for isolating the ATPase complex of rat liver mitochondria in a form that is detergent soluble and sensitive to all inhibitors of the terminal steps of oxidative phosphorylation. The chapter discusses analytical procedures, which include ATPase assays, inhibitor assays, and protein determinations. Preparation of phosphate- washed inner membrane vesicles is also discussed. Inner membrane vesicles that exhibit a high ATPase specific activity are used for the isolation of soluble oligomycin-sensitive ATPase.