Partial Purification and Characterization of the Vacuolar H+‐ATPase of Mammalian Synaptic Vesicles

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Abstract
Several major proteins of synaptic vesicles from rat or cow brain sediment as a large complex on sucrose density gradients when solubilized in nonionic detergents. A vacuolar H+-ATPase identified by sensitivity to bafilomycin A1 appears to be associated with this oligomeric protein complex. Two subunits of this complex, synaptic vesicle proteins S and U, correspond to the 57-kDa (B) and 39-kDa accessory (Ac39) subunits, respectively, of bovine chromaffin granule vacuolar H+-ATPase as shown by Western immunoblot analysis. The five subunits of the oligomeric complex constitute .apprx. 20% of the total protein of rat brain synaptic vesicles. Taken together, these results strongly suggest that the abundant, multisubunit complex partially purified from brain synaptic vesicles by density gradient centrifugation is a vascuolar H+-ATPase. Bafilomycin A1 completely blocks proton pumping in rat brain synaptic vesicles as measured by [14C]methylamine uptake and also blocks catecholamine accumulation measured by [3H]dopamine uptake moreover, ATPase activity, [14C]methylamine uptake, and [3H]dopamine uptake are inhibited by bafilomycin A1 at similar I50 values of .apprx. 1.7 nmol/mg of protein. These findings indicate that the vacuolar H+-ATPase is essential for proton pumping as well as catecholamine uptake by mammalian synaptic vesicles.