[34] Preparation and properties of complex V

Complex V is the segment of the mitochondrial oxidative phosphorylation system that is associated with adenosine triphosphate (ATP) synthesis and hydrolysis. In the isolated state, it catalyzes uncoupler- and oligomycin-sensitive ATP–Pi exchange and oligomycin-sensitive ATP hydrolysis. The ATP–Pi exchange reaction involves the exchange of the terminal phosphoryl group of ATP with inorganic [33P] phosphate (33Pi) added to the reaction mixture. The assay involves the determination of the amount of [33P] ATP formed as a function of time and enzyme concentration. This chapter describes the basic features of the purification procedure. All preparative steps are carried out at 0°–4°C. The pH values of buffers, deoxycholate, and cholate, which appear in parentheses throughout the sections on purification and assay methods, are adjusted at room temperature (20°–25°C). Mitochondria from beef heart are suspended in 0.25 M sucrose and stored at −20°C until used. Complex V can be prepared directly from mitochondria or preferably from phosphorylating submitochondrial particles (ETPH).