Partial purification and properties of flyhead cholinesterase

Abstract

Abstract-Houseflyhead 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 pM/hr/mg of organic matter (157 x 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 y0 la-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.

LOCALIZATION OF CHOLINESTERASE ACTIVITY IN HOUSEFLY THORACES: INHIBITION OF CHOLINESTERASE WITH ORGANOPHOSPHATE COMPOUNDS1

“…It seems unlikely that 2 different ChE's would occupy the apparently same histological sites and also demonstrate the same susceptibility to inhibition by different O-P's such as tepp and dicrotophos. The interpretation of these results supports the conclusion of Van Asperen (1959), Dauterman et al (1962), and Chaudhary et al (1966) that, in the housefly and Triboli.m c o n f . s .…”

“…The reverse effect was obtained with a reported selective inhibitor of specific ChE (ACHE). Although housefly ChE has, in general, the characteristic properties of specific ChE (ACHE) ( Van Asperen, 1959;Dauterman et al, 1962), all these data clearly indicate that the ChE of the housefly is less specific than AChE from other sources mentioned.…”

Occurrence and Inheritance of Esterases in Musca Domestica

“…This was performed on microscope slides according to WIEME (1959). The conditions were as described by DAIJTERMAN, TALENS & VAN ASPEREN (1962), except that the electrophoresis time was mostly 12 min. Microscope slides (76)< 26 mm) were covered with a 1.5 mm thick layer of agargel (Difco agar noble 0.9%), made up in veronal buffer (pH 8.6; t~ = 0.05).…”

Species Distribution and Characterization Studies of Sex-Limited Esterases within the virgifera Group of Diabrotica (Coleoptera: Chrysomelidae)

“…A number of protocol variations involving Coomassie Blue R-250 or G-250 (Bio-Rad Laboratories, Richmond, Cal.) were used to stain gels for general protein (Dauterman et al 1962, Diezel et al 1972, Reisner et al 1975, Blakesley & Boezi 1977, Steiner & Joslyn 1979, Reisner 1984. Additionally, protein bands were resolved using variations of a Bio-Rad silver stain kit procedure on gels initially stained with Coomassie Blue G-250 by the method of Steiner & Joslyn (1979).…”