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Proton ATPase of rat liver mitochondria: A rapid procedure for purification of a stable, reconstitutively active F1 preparation using a modified chloroform method
Authors:Noreen Williams  L Mario Amzel  Peter L Pedersen  
Institution:1. Laboratory for Molecular and Cellular Bioenergetics, Department of Physiological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 USA;2. Department of Biophysics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 USA
Abstract: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.
Keywords:proton ATPase  F1-ATPase  ATP synthase  ATPase  F0F1-ATPase  mitochondrial ATPase
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