Carroll_1994_Brain.Res_633_112

Some of the choline-O-acetyltransferase (EC 2.3.1.6; ChAT) in rat hippocampal nerve terminals is non-ionically associated with membranes. The intent of the present report was to ascertain whether any of this membrane-bound ChAT might be associated with synaptic vesicles. To test this possibility, synaptosomal (P2) fractions were hypo-osmotically shocked in water, salt washed to remove ionically-bound ChAT, subjected to sucrose density gradient centrifugation, and the activity of ChAT compared with the amount of occluded ACh in the various subcellular fractions. A peak of ChAT and occluded ACh occurred in that fraction of the gradient (0.4 M sucrose) acknowledged to be enriched in synaptic vesicles. In other experiments, Immunobeads coated with an antibody directed against the synaptic vesicle specific SV2 protein immunoprecipitated both ChAT and occluded ACh from the 0.4 M sucrose fraction, but no other fraction. Immunobeads coated with an anti-ChAT antiserum immunoprecipitated synaptophysin from the 0.4 M sucrose fraction, an effect which was blocked by pretreatment of the anti-ChAT Immunobeads with purified ChAT. These results suggest that some of the membrane-bound ChAT in rat hippocampal nerve terminals is associated with cholinergic synaptic vesicles.