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Membrane-bound choline acetyltransferase from human brain: Purification and properties

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Abstract

Choline acetyltransferase (ChAT; EC 2.3.1.6) was separated from human caudate/putamen into three fractions by successive extractions into apotassium 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 μmol 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 of 10% SDS gel.

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Authors and Affiliations

  1. Kinsmen Laboratory of Neurological Research Department of Psychiatry, University of British Columbia, 2255 Westbrook Mall, V6T 1W5, Vancouver, B. C., Canada

    J. H. Peng, P. L. McGeer & E. G. McGeer

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  1. J. H. Peng
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  2. P. L. McGeer
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  3. E. G. McGeer
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Special Issue dedicated to Prof. Eduardo De Robertis.

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Peng, J.H., McGeer, P.L. & McGeer, E.G. Membrane-bound choline acetyltransferase from human brain: Purification and properties. Neurochem Res 11, 959–971 (1986). https://doi.org/10.1007/BF00965586

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  • DOI: https://doi.org/10.1007/BF00965586

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