58] Preparation and reconstitution of F1F0 and F0 from Escherichia coli

Abstract

This chapter discusses the preparation and reconstitution of F1F0 and F0 from Escherichia coli. The ATP synthase, F1F0, is extracted from everted membrane vesicles of E. coli by detergent and purified by ion-exchange chromatography and precipitation with polyethylene glycol (PEG). The proton conductor, F0, is isolated from F1F0 by dissociation of F1. The enzymatic activities of F1F0 assayed are ATPase, ATP-32Pi exchange, and ATP-dependent quenching of acridine dye fluorescence. F0 is tested for proton conduction by direct pH electrode measurements and by quenching of acridine dye fluorescence. These activities are inhibited by dicyclohexylcarbodiimide (DCCD). The easiest assay is the assay of ATPase activity, as it is not disturbed by detergents as long as some residual phospholipid is present. The other activities are dependent on a vesicular structure, so proteoliposomes have to be reconstituted from the enzyme preparation and phospholipids.