Reconstitution of Mitochondrial Oligomycin and Dicyclohexylcarbodiimide‐Sensitive ATPase

1. Oligomycin and dicyclohexylcarbodiimide‐sensitive ATPase was isolated from beef‐heart mitochondria and treated with 3.5 M NaBr in order to remove F 1 . The residue, called F 0 , was found to consist of seven components. Five of these are stained by Coomassie blue after dodecyl‐sulfate‐polyacrylamide‐gel electrophoresis. Two of them correspond to the oligomycin‐sensitivity‐conferring protein and coupling factor F 6 , with apparent molecular weights of 21000 and 9400, respectively. Three additional polypeptides of molecular weights 23000, 10500 and 8600 were not identified with known proteins. Two components not stained with Coomassie blue were detected by autoradiography of the gels of F 0 preincubated with [ 14 C]dicyclohexylcarbodiimide. These two components probably represent monomeric and oligomeric forms of the dicyclohexylcarbodiimide‐binding protein. 2. F 0 induced an oligomycin and dicyclohexylcarbodiimide‐sensitive enhancement of K + + valinomycin‐driven proton translocation across the membrane of artificial phospholipid vesicles. 3. The interaction of F 0 with purified, soluble beef heart F 1 was investigated. F 0 was capable of binding F 1 and conferring oligomycin and dicyclohexylcarbodiimide sensitivity and cold stability on its ATPase activity. Furthermore F 0 was found to diminish the specific activity of F 1 ‐ATPase. A comparison of these effects at varying F 0 /F 1 ratios shows that F 0 binds F 1 in both an oligomycin‐sensitive and an oligomycin‐insensitive manner, and that both types of binding involve a conferral of cold stability and a decrease in specific activity. High F 0 /F 1 ratios favoured the oligomycin‐sensitive type of binding, indicating that F 1 binds preferentially to oligomycin‐sensitivity‐conferring sites. Treatment of ATPase complex with trypsin resulted in an F 0 with a decreased proportion of oligomycin‐sensitivity‐conferring binding sites and a diminished ability to lower the specific activity and cold lability of F 1 . 4. Reconstitution of F 0 treated with trypsin and F 1 , oligomycin‐sensitivity‐conferring protein and F 6 showed that at a constant amount of F 1 bound, both oligomycin‐sensitivity‐conferring protein and F 6 increased the oligomycin sensitivity of ATPase activity. It was therefore concluded that both of these coupling factors are involved in the conferral of oligomycin sensitivity. 5. The effect of the order of addition of F 1 , oligomycin‐sensitivity‐conferring protein and F 6 to F 0 on the reconstitution of oligomycin‐sensitive ATPase activity, and of F 1 and oligomycin‐sensitivity‐conferring protein to submitochondrial particles on the reconstitution of respiratory control, was investigated. The highest values of oligomycin sensitivity and respiratory control were obtained when F 1 was added as the first component, indicating that F 1 plays a directing role in the organisation of the components.