Characterization of a Mg2+-ATPase and a proton pump in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata

Abstract

Cholinergic synaptic vesicles from the electric organ of Torpedo marmorata are associated with a Mg2+-ATPase insensitive to ouabain and oligomycin. Treatment of vesicle membranes with dichloromethane releases a Mg2+-ATPase with apparent molecular mass of around 250 kDa [kilodaltons] as determined by gel filtration. The vesicular ATPase resembles the mitochondrial F1-ATPase in these properties. Gel electrophoresis of the solubilized ATPase shows however that only a single 50-kDa band is present as compared to the .alpha.-subunit (52 kDa) and .beta.-subunit (50 kDa) of electric organ mitochondrial F1-ATPase present in this range of molecular mass range. In agreement, covalent photoaffinity labeling of isolated vesicles with azido-ATP shows a 50-kDa band. Vesicle ghosts accumulated [14C]methylamine in an ATP-dependent manner indicating the presence of an inwardly directed proton pump. Cholinergic vesicles contain a proton pump probably driven by the Mg2+-ATPase here described, which generates an electrochemical gradient across the vesicle membrane and is necessary for uptake and storage of acetylcholine within the vesicles.