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 F1. The residue, called F0, was found to consist of seven components. Five of these are stained by Coomassie blue after dodecylsulfate-polyacrylamide-gel electrophoresis. Two of them correspond to the oligomycin-sensitivity-conferring protein and coupling factor F6, with apparent molecular weights of 21,000 and 9,400, respectively. Three additional polypeptides of molecular weights 23,000, 10,500 and 8,600 were not identified with known proteins. Two components not stained with Coomassie blue were detected by autoradiography of the gels of F0 preincubated with [14C]dicyclohexylcarbodiimide. These two components probably represent monomeric and oligomeric forms of the dicyclohexylcarbodiimide-binding protein. 2. F0 induced an oligomycin and dicyclohexylcarbodiimide-sensitive enhancement of K+ + valinomycin-driven proton translocation across the membrane of artificial phospholipid vesicles. 3. The interaction of F0 with purified, soluble beef heart F1 was investigated. F0 was capable of binding F1 and conferring oligomycin and dicyclohexylcarbodiimide sensitivity and cold stability on its ATPase activity. Furthermore F0 was found to diminish the specific activity of F1-ATPase. A comparison of these effects at varying F0/F1 ratios shows that F0 binds F1 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 F0/F1 ratios favoured in oligomycin-sensitive type of binding, indicating that F1 binds preferentially to oligomycin-sensitivity-conferring sites. Treatment of ATPase complex with trypsin resulted in an F0 with a decreased proportion of oligomycin-sensitivity-conferring binding sites and a diminished ability to lower the specific activity an cold lability of F1. 4. Reconstitution of F0 treated with trypsin and F1, oligomycin-sensitivity-conferring protein and F6 showed that at a constant amount of F1 bound, both oligomycin-sensitivity-conferring protein and F6 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 F1, oligomycin-sensitivity-conferring protein and F6 to F0 on the reconstitution of oligomycin-sensitive ATPase activity, and of F1 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 F1 was added as the first component, indicating that F1 plays a directing role in the organisation of the components.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001965:Bromides; D002234:Carbodiimides / Q000494:pharmacology*; D002352:Carrier Proteins; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D008081:Liposomes / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases; D008970:Molecular Weight; D009840:Oligomycins / Q000494:pharmacology*; D010086:Oxidative Phosphorylation Coupling Factors; D006180:Proton-Translocating ATPases; D011522:Protons; D012964:Sodium; D017670:Sodium Compounds
D001965:Bromides; D002234:Carbodiimides; D002352:Carrier Proteins; D008081:Liposomes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009840:Oligomycins; D010086:Oxidative Phosphorylation Coupling Factors; D011522:Protons; D017670:Sodium Compounds; D004024:Dicyclohexylcarbodiimide; D012964:Sodium; D000251:Adenosine Triphosphatases; C000628217:F(6) ATPase; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein; C027938:sodium bromide