Preparative Nondenaturing Gel Electrophoresis Used in the Purification of an Esterase Involved in Insecticide Resistance, Rev C ( Dr Albert J Ketterman an...)

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Preparative Nondenaturing Gel Electrophoresis Used in the Purification of an Esterase Involved in Insecticide Resistance, Rev C

Dr Albert J Ketterman and SHP Parakrama Karunaratne, Dept. of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel St, London WC1E 7HT UK

Introduction
In attempts to control Culex quinquefasciatus as a worldwide vector of diseases such and filariasis and encephalitis, mosquito field populations are sprayed with organophosphate insecticides. However, mosquitoes develop tolerance to organophosphate.

One of the mechanisms of organophosphate resistance involves either a different form and/or increased amounts in a carboxyolesterase (EC 3.1.1.1). Carboxylesterase A₂ is one of two enzymes found elevated together in Culex mosquitoes. The putative mechanism involving the resistance-conferring carboxylesterase A₂ is an increased expression in this enzyme for the sequestration of, and cross-resistance to, various organophosphate and carbamate insecticides (Raymond et al. 1991).

The question of a qualitative change in the carboxylesterase has not been fully explored with a purified enzyme. Here we report the use of Bio-Rads Model 491 prep cell for preparative, nondenaturing gel electrophoresis as a final step in purifying the native carboxylesterase A₂.

Methods
Carboxylesterase A₂ was isolated from the supernatant of a mosquito homogenate by sequential ion exchange, hydrophobic interaction, and hydroxyapatite chromatography, followed by a final purification step using the Model 491 prep cell (Ketterman et al. 1992).

Preparative Native PAGE
As suggested in the Model 491 prep cell instruction manual, the gel composition (monomer concentration) for maximum resoluti
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( Dr Albert J Ketterman an...)Preparative Nondenaturing Gel Electrophoresis Used in the Purification of an Esterase Involved in Insecticide Resistance, Rev C