Partial purification and characterization of the vacuolar H(+)-ATPase of mammalian synaptic vesicles.
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 approximately 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 vacuolar 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 approximately 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.
Floor
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Department of Anatomy, University of Wisconsin Medical School, Madison.
Leventhal
P S
PS
Schaeffer
S F
SF
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001921:Brain / Q000201:enzymology*; D004298:Dopamine / Q000493:pharmacokinetics; D018942:Macrolides; D008565:Membrane Proteins / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000378:metabolism; D051381:Rats; D013572:Synaptic Vesicles / Q000201:enzymology* / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology*
D000900:Anti-Bacterial Agents; D018942:Macrolides; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C057620:bafilomycin A; D000251:Adenosine Triphosphatases; D004298:Dopamine