PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM OX BRAIN STRIATE NUCLEI

Abstract—

• 1 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.
• 2 The enzyme was found to be a stable and relatively basic protein, with a molecular weight of 65,000.
• 3 In the catalysed reactions, , k₁, was about four times k₂, 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).
• 4 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.