ISOLATION AND CHARACTERIZATION OF AN INSECTICIDE-RESISTANCE-ASSOCIATED ESTERASE IN THE TOBACCO BUDWORM HELIOTHIS-VIRESCENS (F)

1-Naphthyl acetate (1-NA) esterase activity was elevated in whole body homogenates of first, third, and fifth stadium tobacco budworms resis tant to thiodicarb and cypermethrin compared to susceptible budworms a t the same developmental stage. Increased esterase activity was attrib uted mostly to three esterases designated A1, B1, and C1 with isoelect ric points of 4.8, 5.1, and 5.3, respectively, as determined by isoele ctric focusing-polyacrylamide gel electrophoresis (IEF-PAGE). These th ree esterases were purified by preparative liquid column isoelectric f ocusing followed by slab IEF-PAGE and sodium dodecyl sulfate-PAGE. Spe cific antibody to the resistance-associated esterase of the house fly (anti-E1, N. Motoyama) and the green peach aphid (anti-E4, A. Devonshi re) was immunoreactive on Western blots with purified esterase A1, whi le minimal or no reactivity was noted for esterases B1 and C1. Anti-E4 detected A1 in 10,000g supernatant from homogenates of resistant and susceptible tobacco budworms. Kinetic studies demonstrated that methyl paraoxon, cypermethrin, DDT, and thiodicarb were competitive inhibito rs of the 1-NA esterase activity of A1, whereas eserine hemisulfate ha d no effect on activity. An amino-terminal sequence analysis revealed that residues Gly-12, Arg-15, and Gly-16 were conserved with Culex est erase B1 (CuB1), Drosophila melanogaster esterase-6 (DmE6) and acetylc holinesterase, H. virescens JH esterase, human butyrylcholinesterase, and rabbit liver esterase form 2, while Arg-15 was missing in E4 from Myzus persicae. A1 demonstrated the greatest sequence homology with th at of the DmE6 of Drosophila and the resistance-associated esterase, C uB1, of Culex. (C) 1995 Academic Press, Inc.