Proton ATPase of rat liver mitochondria: a rapid procedure for purification of a stable, reconstitutively active F1 preparation using a modified chloroform method.
A method is described for the purification of rat liver F1-ATPase by a modification of the chloroform extraction procedure originally described by Beechey et al. (Biochem. J. (1975) 148, 533). Purified liver membrane vesicles are extracted with chloroform in the presence of ATP and EDTA. The procedure yields pure F1 in only 2-3 h without the necessity of ion-exchange chromatography. The enzyme exhibits the alpha, beta, gamma, delta, and epsilon bands characteristic of F1-ATPase. It has a high ATPase specific activity, and is reconstitutively active, catalyzing high rates of ATP synthesis. Significantly, it can be readily crystallized. If desired, the enzyme can be passed over a gel filtration column to place it in a stabilizing phosphate-EDTA buffer, lyophilized and stored indefinitely at -20 degrees C.
D000818:Animals; D002725:Chloroform; D003080:Cold Temperature; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000201:enzymology; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000302:isolation & purification*; D051381:Rats; D013367:Submitochondrial Particles / Q000201:enzymology
D002725:Chloroform; D006180:Proton-Translocating ATPases