Large-scale chromatographic purification of F1F0-ATPase and complex I from bovine heart mitochondria.
A new chromatographic procedure has been developed for the isolation of F1F0-ATPase and NADH:ubiquinone oxidoreductase (complex I) from a single batch of bovine heart mitochondria. The method employed dodecyl beta-delta-maltoside, a monodisperse, homogeneous detergent in which many respiratory complexes exhibit high activity, for solubilization and subsequent purification by ammonium sulphate fractionation and column chromatography. A combination of anion-exchange, gel-filtration, and dye-ligand affinity chromatography was used to purify both complexes to homogeneity. The F1F0-ATPase preparation contains only the 16 known subunits of the enzyme. It has oligomycin-sensitive ATP hydrolysis activity and, as demonstrated elsewhere, when reconstituted into lipid vesicles it is capable of ATP-dependent proton pumping and of ATP synthesis driven by a proton gradient [Groth and Walker (1996) Biochem. J. 318, 351-357]. The complex I preparation contains all of the subunits identified in other preparations of the enzyme, and has rotenone-sensitive NADH:ubiquinone oxidoreductase and NADH:ferricyanide oxidoreductase activities. The procedure is rapid and reproducible, yielding 50-80 mg of purified F1F0-ATPase and 20-40 mg of purified complex I from 1 g of mitochondrial membranes. Both preparations are devoid of phospholipids, and gel filtration and dynamic light scattering experiments indicate that they are monodisperse. Therefore, the preparations fulfil important prerequisites for structural analysis.
Buchanan
S K
SK
Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
Walker
J E
JE
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000302:isolation & purification; D003576:Electron Transport Complex IV / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000201:enzymology*; D008970:Molecular Weight; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry / Q000302:isolation & purification*; D010743:Phospholipids / Q000032:analysis; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012542:Scattering, Radiation
D003902:Detergents; D005960:Glucosides; D010743:Phospholipids; C040358:dodecyl maltoside; D016660:NAD(P)H Dehydrogenase (Quinone); D003576:Electron Transport Complex IV; D006180:Proton-Translocating ATPases; D014450:Electron Transport Complex III