Purification and Some Properties of Choline Acetyltransferase from Catfish Brain

Choline acetyltransferase (CAT) from catfish brain has been purified to electrophoretic homogeneity by a combination of ammonium sulfate fractionation, gel filtration, and preparative polyacrylamide gel electrophoresis. The purified enzyme solutions showed only one protein band on both 7.5% polyacrylamide gel and gradient (3.5% to 15%) polyacrylamide gel with the enzyme activity coincident with the protein band. Furthermore, when the gel slice from which the enzyme was extracted was applied to a gradient polyacrylamide gel, a single protein band was also obtained. The purified enzyme preparations represented a 1510‐fold purification over the crude homogenate. The moiecular weight of the enzyme was estimated as 128.000 and 140,000 from gel filtration and gradient (3.5% to 15%) polyacrylamide gel, respectively. On sodium dodecyl sulfate‐gel, the purified enzyme preparations showed two protein bands, with molecular weights of 34,000 and 68,000. Since the native enzyme may be a tetramer, the 68.000 subunit may be a dimer of the smallest subunit. The maximum activity of the enzyme appears at pH 7.2; the values of Km for choline and for acetyl CoA are 400 and 92 μM, respectively, and Vmax was obtained as 2.2 × 10‐3μmol/min/mg protein. CAT antibodies were obtained from rabbit that had received six biweekly injections with a total of 120 μg of the purified protein. Double immunodiffusion test with CAT antibodies and a crude enzyme extract from catfish brains showed only a single, sharp precipitin band, suggesting that the antibodies are specific only to CAT and antigen and that the purified CAT preparation is monodisperse.