CDNA CLONING, BACULOVIRUS-EXPRESSION AND KINETIC-PROPERTIES OF THE ESTERASE, E3, INVOLVED IN ORGANOPHOSPHORUS RESISTANCE IN LUCILIA-CUPRINA

Resistance to organophosphorus insecticides (OPs) in the sheep blowfly , Lucilia cuprina, is associated with a non-staining phenotype of the carboxylesterase isozyme, E3 (E.C. 3.1.1.1). Here, we show that a memb er of alpha-esterase multigene family, Lc alpha E7, encodes E3. An Lc alpha E7 cDNA has been isolated from an OP-susceptible strain and expr essed in a baculovirus. The expressed product is the same as E3 in its electrophoretic mobility and preference for alpha-over beta-naphthyl acetate as substrate, Its preference (k(cat)/K-m) for a range of carbo xylester substrates is alpha-naphthyl butyrate>alpha-naphthyl propiona te>alpha-naphthyl acetate>methylthiobutyrate>p-nitrophenyl acetate, Th e enzyme is potently inhibited by OPs (k(i) [paraoxon] = 6.3 +/- 1.4 x 10(7)/M/min, k(i) [chlorfenvinphos] = 5.9 +/- 0.6 x 10(7)/M/min) and exhibits a high turn-over of methylthiobutyrate (1009/s), consistent w ith its proposed homology to the ali-esterase that is thought to mutat e to confer OP resistance in Musca domestica. E3 shares 64% amino acid identity with its Drosophila melanogaster homologue, Dm alpha E7, and is also closely related to other esterases involved in OP resistance such as the B1 esterase of Culex pipiens (38%) and E4 of Myzus persica e (30%). (C) 1997 Elsevier Science Ltd.