Choline Acetyltransferase from the electric organ of Electrophorus electricus (L.)--physicochemical characterization and immunochemical identification.
It is well known that the regulation of choline acetyltransferase (ChAT) activity, under physiological conditions, is important for the development and neuronal activities of cholinergic systems. The purification of ChAT has been obtained from many sources such as electric organs of fishes, Drosophila melonogaster, and mammals. We have prepared choline acetyltransferase from a pool of supernatants obtained by differential centrifugation of electric organ homogenates from Electrophorus electricus (L.) in Tris-phosphate buffer, 0.05 M, pH 7.6. The first step of the enzyme purification was performed by ammonium sulfate precipitation at 40% and 80%. The precipitate at 80% was solubilized with sodium-phosphate buffer 0.05 M, pH 7.6, dialyzed, chromatographed on DEAE-52 column and the active fraction submitted to FPLC system columns (Mono-Q: ion exchange- Superose-12: gel filtration). ChAT activity from the eluates was estimated by Fonnun's method [Fonnun, 1975], with Acetyl-Coenzyme A tritium labelled ([3H]AcCoA) as substrate, and the synthesis of 3HACh formed was measured. The peak from gel filtration showed a relative molecular mass of 80 offkDa with highest activity in the order of 77,42 nmoles ACh/min/mg protein. This fraction was analyzed by SDS-PAGE and a band of 42 kDa was detected with Coomassie blue stain, indicating that the enzyme is formed by two subunits. Employing an antibody, the presence of ChAT was confirmed with the Western blotting technique. Isoelectrofocusing analysis demonstrated two isoforms with pI of 6,49 and 6,56, respectively.
Laboratório de Físico-Química Biológica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil.
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Tavares
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Hassón-Voloch
D000818:Animals; D015153:Blotting, Western; D002795:Choline O-Acetyltransferase / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004557:Electric Organ / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004593:Electrophorus; D007202:Indicators and Reagents; D008970:Molecular Weight
D007202:Indicators and Reagents; D002795:Choline O-Acetyltransferase