title: Comparison of an Esterase Associated with Organophosphate Resistance in Lucilia cuprina with an Orthologue Not Associated with Resistance in Drosophila melanogaster
abstract: Orthologous E3 and EST23 carboxylesterases have been enriched over 200-fold from organophosphate (OP) susceptible strains of Lucilia cuprina and Drosophila melanogaster, respectively. Mutants of E3 are associated with OP resistance but no resistance mutations of EST23 are known. The behaviours of the two enzymes were very similar during purification which involved differential centrifugation followed by three or four ion exchange and gel filtration chromatographic steps. Nondenaturing polyacrylamide gel electrophoresis and histochemical staining for esterase activity revealed no other esterases in the enriched material. Two-dimensional polyacrylamide gel electrophoresis (native followed by denaturing) showed that a major 70-kDa component of each preparation comigrates with E3 and EST23 activities, respectively. Kinetic properties of the enzymes are also very similar. Estimates of Km, Kcat, and Kcat/Km for alpha-naphthyl acetate are 42 ± 18 &mu;M, 19 sec-1, and 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 are potently inhibited by dibrom and less potently by another OP, diisopropylflurophosphate. E3 is also potently inhibited by paraoxon, whereas EST23 is at least 8-fold less susceptible to inhibition by paraoxon. This supports previous analyses of crude homogenates which showed that E3 is more susceptible to inhibition by paraoxon and fenitrooxon than is EST23 or the target site for OP action, acetylcholinesterase. It is proposed that the unusual affinity of E3 for such OPs is a necessary precondition for mutations that enable it to confer OP resistance on L. cuprina.
authors.affiliation: CSIRO Division of Entomology, Canberra, ACT 2601, Australia
authors.initials: AG
authors.lastname: Parker
authors.initials: PM
authors.lastname: Campbell
authors.initials: ME
authors.lastname: Spackman
authors.initials: RJ
authors.lastname: Russell
authors.initials: JG
authors.lastname: Oakeshott