title: Membrane-bound choline acetyltransferase from human brain: purification and properties.
abstract: Choline acetyltransferase (ChAT; EC 2.3.1.6) was separated from human caudate/putamen into three fractions by successive extractions into a potassium phosphate buffer, a high salt (NaCl) buffer and a buffer containing 0.6% Triton X-100. The Triton-X-solubilized fraction is the membrane-bound ChAT (mChAT) and represents about 40% of the total ChAT. After centrifugation, mChAT was precipitated by ammonium sulfate at 35-65% saturation. The crude enzyme preparation was fractionated in turn on a DEAE-Sepharose, a hydroxylapatite and a phosphocellulose columns. Finally, mChAT was applied to a CoA-Sepharose column equilibrated with buffer containing 100 mM choline chloride and was specifically eluted with buffer containing acetyl-CoA. The presence of both substrates greatly stabilized the enzyme and ChAT was recovered almost quantitatively. The final preparation of mChAT has a specific activity of 37.2 mumol of acetylcholine synthesized per min-mg protein. The purified mChAT has a pH optimum of 8.3. It migrated as two bands on SDS-PAGE with molecular weights of 67,000 and 62,000 daltons, respectively. Immunoblot autoradiography showed that an antiserum prepared previously against soluble ChAT also cross-reacted with both bands of mChAT, indicating that both forms of this enzyme are related. Furthermore, as previously reported for soluble ChAT, Fab-Sepharose chromatography could be used for the purification of mChAT and this preparation also resolved into two bands on 10% SDS gel.
mesh_terms: D002421:Caudate Nucleus / Q000201:enzymology*; D002795:Choline O-Acetyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008566:Membranes / Q000201:enzymology; D011699:Putamen / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D002795:Choline O-Acetyltransferase