79] Preparation and properties of a factor conferring oligomycin sensitivity (F0) and of oligomycin-sensitive ATPase (CF0·F1)

Abstract

This chapter examines the preparation and properties of a factor conferring oligomycin sensitivity (F 0 ) and of oligomycin- sensitive ATPase (CF 0 ·F 1 ). The assay of F 0 activity is based on the inhibition of ATPase activity of coupling factor 1 (F 1 ) by oligomycin in the presence of F 0 . The ATPase activity of purified coupling factor 1 is not inhibited by oligomycin. After adsorption to F 0 , the ATPase activity becomes sensitive to oligomycin and to certain other energy-transfer inhibitors. In the preparation of F 0 , F 0 is incubated in the presence of F 1 (1-2 μ g) and 5 millimicromoles of oligomycin in a final volume of 0.8 ml, containing 0.025 M Tris-sulfate, pH 7.4, at 30° for 5 minutes. The assay of preparations of CF 0 and CF 0 ·F 1 is similar to that of F 0 except that F 1 was incubated with 100 μ g of CF 0 protein for 3 minutes prior to the addition of 0.01 ml of the 1% phospholipid suspension. F 0 preparations contain the entire respiratory chain and have phospholipid content, which is slightly higher than that of submitochondrial particles. On the other hand, preparations of CF 0 and CF 0 ·F 1 have no DPNH or succinate oxidase activity and contain little phospholipid, but they are capable of binding about 1 micromole of phospholipid phosphorus per milligram of protein.