PURIFICATION OF CHOLINE-ACETYLTRANSFERASE FROM RAT AND COW BRAIN

Choline acetyltransferase was extensively purified from rat cerebra and bovine caudate nuclei. The initial steps in each purification were essentially those described by Malthe-Sørenssen et al. [Malthe-SøSrenssen, D., Eskeland, T., & Fonnum, F. (1973) Brain Res. 62, 517-522]. Rat cerebral choline acetyltransferase was solubilized, fractionated by precipitation with acetic acid and with ammonium sulfate, and chromatographed on carboxymethyl-Sephadex C-50. Further purification was obtained by sequential chromatography on an affinity column of coenzyme A (CoA)-Sepharose and on hydroxylapatite to yield a final product with a specific activity of 40 μmol min-1 mg-1. The yield was 14%, with an overall purification of 18 300-fold. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme was 68% pure. The first three steps of the purification of rat choline acetyltransferase were used in an identical manner for purification of the enzyme from bovine caudate nuclei. In this case, chromatography on carboxymethyl-Sephadex C-50 resulted in the separation of two peaks of enzyme activity. In order of their elution, these peaks accounted for 75-80% (Bov I) and 20-25% (Bov II) of the eluted activity. Purification of Bov I by using affinity chromatography on CoA-Sepharose and chromatography on hydroxylapatite generated a final product with a specific activity of 58.8 μmol min-1 mg-1. A yield of 18% was obtained with an overall purification of 21 500-fold. The final preparation was estimated to be 80% pure. Bov II was purified further only by affinity chromatography. The specific activities of these preparations are the highest yet reported for choline acetyltransferase from a mammalian source. Although highly purified, the enzyme is not yet homogeneous. The findings are discussed with respect to the results obtained by other investigators, some of whom claim to have purified choline acetyltransferase to homogeneity. © 1979, American Chemical Society. All rights reserved.