Open AccessCharacterization of a Mg 2+ ‐ATPase and a proton pump in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata
Author(s) -
HARLOS Peter,
LEE Deborah Anne,
STADLER Herbert
Publication year - 1984
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1984.tb08485.x
Subject(s) - vesicle , synaptic vesicle , torpedo , atpase , oligomycin , biochemistry , biophysics , ouabain , cholinergic , electrochemical gradient , chemistry , biology , membrane , acetylcholine receptor , sodium , enzyme , endocrinology , receptor , organic chemistry
Cholinergic synaptic vesicles from the electric organ of Torpedo marmorata are associated with a Mg 2+ ‐ATPase insensitive to ouabain and oligomycin. Treatment of vesicle membranes with dichloromethane releases a Mg 2+ ‐ATPase with apparent molecular mass of around 250 kDa as determined by gel filtration. The vesicular ATPase resembles the mitochondrial F 1 ‐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 a‐subunit (52 kDa) and β‐subunit (50 kDa) of electric organ mitochondrial F 1 ‐ATPase present in this range of molecular mass range. In agreement, covalent photoaffinity labelling of isolated vesicles with azido‐ATP shows a 50‐kDa band. Vesicle ghosts were found to accumulate [ 14 C]methylamine in an ATP‐dependent manner indicating the presence of an inwardly directed proton pump. We conclude that cholinergic vesicles contain a proton pump probably driven by the Mg 2+ ‐ATPase here described, which generates an electrochemical gradient across the vesicle membrane and is necessary for uptake and storage of acetylcholine within the vesicles.