Proton ATPase of rat liver mitochondria: A rapid procedure for purification of a stable, reconstitutively active F 1 preparation using a modified chloroform method

Abstract

A method is described for the purification of rat liver F 1-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 F 1 in only 2–3 h without the necessity of ion-exchange chromatography. The enzyme exhibits the α, β, γ, δ, and ϵ bands characteristic of F 1-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°C.