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

• Published in: Biochemical journal 1980-11, Vol.192 (2), p.483-488
• Main Authors: Kozlov, I A, Milgrom, Y M, Tsybovski, I S
• Format: Article
• Language: English
• Subjects: Adenine Nucleotides - pharmacology; Adenosine Triphosphatases - antagonists & inhibitors; Adenosine Triphosphatases - isolation & purification; Adenosine Triphosphatases - pharmacology; Animals; Benzoates - pharmacology; Binding Sites; Cattle; Chemical Phenomena; Chemistry; Electrophoresis, Polyacrylamide Gel; Enzyme Precursors - isolation & purification; Intracellular Membranes - enzymology; Lithium; Methods; Mitochondria - enzymology; Mitochondria, Heart - enzymology; Oligomycins - pharmacology; Proton-Translocating ATPases; Submitochondrial Particles - enzymology; Xylenes - pharmacology
• ISSN: 0264-6021; 0306-3283; 1470-8728
• DOI: 10.1042/bj1920483

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.