Isolation of alpha-subunits of factor F1 from submitochondrial particles and the reconstitution of active ATPase from isolated alpha-subunits and beta-subunits bound to the mitochondrial membrane.
The alpha-subunits of factor-F1 ATPase are removed by extraction of submitochondrial particles with 1.75 M-LiCl, with the consequent loss of ATPase activity. ATPase activity is reconstituted by incubation of LiCl-extracted particles with purified alpha-subunits, and the reconstituted ATPase activity is oligomycin-sensitive. Reconstitution is enhanced by maintenance of the alpha-subunits in reduced form by dithiothreitol or NaBH4 and by modification of the alpha-subunits by p-chloromercuribenzoate, iodoacetic acid or N-ethylmaleimide. Experiments with the mixed anhydride of ATP and mesitylene-carboxylic acid, which was previously shown to interact with the F1 active site, localized on the beta-subunits, indicate that the active site of ATPase is shielded by the alpha-subunits.
D000227:Adenine Nucleotides / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D000818:Animals; D001565:Benzoates / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000302:isolation & purification*; D007425:Intracellular Membranes / Q000201:enzymology; D008094:Lithium; D008722:Methods; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009840:Oligomycins / Q000494:pharmacology; D006180:Proton-Translocating ATPases; D013367:Submitochondrial Particles / Q000201:enzymology*; D014992:Xylenes / Q000494:pharmacology
D000227:Adenine Nucleotides; D001565:Benzoates; D004792:Enzyme Precursors; D009840:Oligomycins; D014992:Xylenes; C019471:mesitylenecarboxylic acid; D008094:Lithium; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases