ATP SYNTHASE FROM BOVINE HEART-MITOCHONDRIA - RECONSTITUTION INTO UNILAMELLAR PHOSPHOLIPID-VESICLES OF THE PURE ENZYME IN A FUNCTIONAL-STATE

A highly purified and monodisperse preparation of proton-translocating F1F0-ATPase from bovine heart mitochondria is an assembly of 16 unlik e polypeptides. This preparation has been reconstituted in the presenc e of various detergents into unilamellar phospholipid vesicles. Incorp oration of the enzyme into vesicles increases the ATP hydrolase activi ty of the enzyme by 10-20-fold, depending on the detergent, and the hi ghest activities of ATP hydrolysis, 70 units/mg, were obtained by reco nstitution from dodecylmaltoside or CHAPS. This activity is mostly sen sitive to inhibitors that act on the F-0 membrane sector of the comple x. From the quenching of the pH-sensitive probe, 9-amino-6-chloro-2-me thoxyacridine, it was shown that the reconstituted enzyme was able to form a transmembrane proton gradient in an ATP-dependent manner. By co -reconstitution of the enzyme with bacteriorhodopsin, it was demonstra ted that in the presence of a light-induced proton gradient the enzyme can synthesize ATP from ADP and phosphate. Therefore, the characteris tic biological functions of the F1F0-ATPase in mitochondria have been demonstrated with the purified enzyme. Thus, in terms of both its phys ical and biochemical properties, the purified enzyme fulfils important pre-requisites for formation of two- and three-dimensional crystals.