Partial purification and properties of flyhead cholinesterase

Abstract

Housefly head cholinesterase was purified using the following steps: (1) freeze-drying of flyheads, (2) solubilization of the enzyme by butanol extraction, (3) ammonium sulphate precipitation at pH 7, (4) heat denaturation of proteins in the presence of acetylcholine for protection of the cholinesterase, (5) ammonium sulphate fractionation at pH 7 and at pH 6, (6) calcium phosphate gel absorption and elution, and (7) acetone fractionation. The final preparation, a solution in glass-distilled water, hydrolysed acetylcholine at a rate of 1600 μM/hr/mg of organic matter (157 × purification). It proved fairly stable and was used for studying some properties of the enzyme. The substrate specificity did not change much in the course of purification. The purified enzyme differed from purified bovine cholinesterase in that it hydrolysed butyrylcholine, triacetin, and phenylbutyrate at a much higher rate. The evidence strongly points to one single enzyme being responsible for the hydrolysis of all substrates studied, including butyrylcholine. Inhibition experiments with organophosphates indicate a probable turnover number in acetylcholine hydrolysis of about 100,000. Experiments on the influence of organic solvents showed that 2–3% n-butanol increases the enzymic activity on choline esters about 60%, and that n-butanol, acetone, and ethanol all lower the rate of inhibition by an organophosphorus compound (diazoxon). Agar gel electrophoresis at pH 8·4 showed the cholinesterase to migrate, probably together with other proteins still present in the purified preparation, at a speed which is about 0·9 times the speed of human serum albumin.