Abstract
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 (P 2) 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 SV 2 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.
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