COMPARISON OF AN ESTERASE ASSOCIATED WITH ORGANOPHOSPHATE RESISTANCE IN LUCILIA-CUPRINA WITH AN ORTHOLOGUE NOT ASSOCIATED WITH RESISTANCE IN DROSOPHILA-MELANOGASTER

Orthologous E3 and EST23 carboxylesterases have been enriched over 200 -fold from organophosphate (OF) susceptible strains of Lucilia cuprina and Drosophila melanogaster, respectively. Mutants of E3 are associat ed with OP resistance but no resistance mutations of EST23 are known. The behaviours of the two enzymes were very similar during purificatio n which involved differential centrifugation followed by three or four ion exchange and gel filtration chromatographic steps. Nondenaturing polyacrylamide gel electrophoresis and histochemical staining for este rase activity revealed no other esterases in the enriched material. Tw o-dimensional polyacrylamide gel electrophoresis (native followed by d enaturing) showed that a major 70-kDa component of each preparation co migrates with E3 and EST23 activities, respectively. Kinetic propertie s of the enzymes are also very similar. Estimates of K-m, K-cat, and K -cat/K-m for alpha-naphthyl acetate are 42 +/- 18 mu M, 19 sec(-1), an d 4.6 x 10(5) M(-1) sec(-1), respectively, for E3, and 62 +/- 25 mu M, 23 sec(-1), and 3.7 x 10(5) M(-1) sec(-1), for EST23. Both enzymes ar e potently inhibited by dibrom and less potently by another OF, diisop ropylflurophosphate. E3 is also potently inhibited by paraoxon, wherea s EST23 is at least 8-fold less susceptible to inhibition by paraoxon. This supports previous analyses of crude homogenates which showed tha t E3 is more susceptible to inhibition by paraoxon and fenitrooxon tha n is EST23 or the target site fbr OP action, acetylcholinesterase. It is proposed that the unusual affinity of E3 for such OPs is a necessar y precondition for mutations that enable it to confer OP resistance on L. cuprina. (C) 1996 Academic Press, Inc.