57] Proton-conducting portion (F0) from Escherichia coli ATP synthase: Preparation, dissociation into subunits, and reconstitution of an active complex

Abstract

Publisher Summary This chapter discusses the preparation, dissociation into subunits, and reconstitution of an active complex for the proton-conducting portion (F 0 ) from Escherichia coli ATP synthase. The F 0 part of the ATP synthase complex (F 1 F 0 ) of E. coli is composed of three different polypeptides and the complex serves as a proton channel. After reconstitution into liposomes a functional F 0 complex can be tested either by measuring passive proton translocation or by adding back F 1 . Passive proton translocation through F 0 is measured using K+-loaded liposomes. ATPase activity of reconstituted F 1 F 0 complexes is measured by determining the liberated orthophosphate. The chapter outlines the procedure for preparation of F 0 by hydrophobic interaction chromatography. Reconstitution of a functional F 0 complex is only achieved by incorporation of all three kinds of subunits into phospholipid vesicles.