PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM OX BRAIN STRIATE NUCLEI

Abstract

Abstract— Choline acetyltransferase was purified from ox brain striate nuclei by an extraction step at pH 5, cation‐exchange chromatography, fractional precipitation with ammonium sulphate, and chromatography on Sephadex G‐200. The enzyme was obtained free of deacylases and cholinesterases, at specific activities of 01‐0‐3 μmol acetylcholine formed per min per mg protein. The enzyme was found to be a stable and relatively basic protein, with a molecular weight of 65,000. In the catalysed reactions, , k 1, was about four times k 2, and the equilibrium constant was approximately 40. For the forward reaction, the Michaelis constant for each substrate was independent of the concentration of the other (choline = 0‐75 mM; acetyl‐CoA = 10 μM), whereas in the back reaction one substrate increased the affinity for the other (acetylcholine = 0‐75‐5 MM; CoA = 25‐150 μM). CoA inhibited acetylcholine synthesis by competing with acetyl‐CoA (K1, = 16 μM). Acetylcholine slightly inhibited the forward reaction (e.g. 45 per cent in 200 mM) without competing with choline or acetyl‐CoA. These data indicate an ordered reaction mechanism; acetyl‐CoA probably always binds before choline.