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Purification and properties of choline acetyltransferase from chicken brain

University of British Columbia

Choline acetyltransferase (ChAc), the enzyme responsible for the synthesis of acetylcholine (ACh), has been extensively purified from chicken brains. Purification procedures included ammonium sulfate fractionation, DEAE-Sephadex (A-25), hydroxyapatite, Sephadex G-150 column chromatography, and affinity chromatography on agarose-hexane-Coenzyme A column. ChAc activity was measured radiochemically. Due to the instability of the enzyme in the course of purification, the most active fraction obtained after agarose-hexane-Coenzyme A chromatography showed a specific activity of only 0.34 μmoles ACh formed/ min./mg protein which corresponded to a 773 fold purification from homogenate. However, on non-denaturing polyacrylamide gel electrophoresis at pH 8.8, the highly purified ChAc preparation showed two distinct bands, and ChAc activity was recovered by slicing and assaying the gel. ChAc activity corresponded to the position of the faster moving band. The same preparation showed one major band and two minor bands on SDS gel electrophoresis. The estimated MW of chicken brain ChAc by gel filtration and SDS gel electrophoresis was 42,500 daltons and no subunit was observed. Two forms of chicken brain ChAc with different Km values for the substrates were eluted from agarose-hexane-CoA column. The pH optimum was estimated to be between pH 7.6-8.0. NaCl, KC1, Ca²⁺ and EDTA stimulated, while Cu²⁺, N-ethylmaleimide and CoA inhibited the enzyme preparation. The apparent Km values for acetyl-CoA and choline were, studied and were found to be similar to those of other mammalian species. The ChAc preparation also showed species specificity by the Ouchterlony double immuno diffusion test. Effect and mechanisms of salt and EDTA activation of ChAc activity are discussed.

Text

2010-03-03

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http://hdl.handle.net/2429/21410

Neuroscience

University of British Columbia

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