Purification and characterization of a phosphorotriester hydrolase from methyl parathion-resistant Heliothis virescens

Abstract

The enzyme that hydrolyzes methyl paraoxon in a methyl parathion-resistant strain of Heliothis virescens was characterized using methyl paraoxon as a substrate. It was localized mainly in the soluble fraction, total activity being highest in the integument/muscle, followed by the fat body and the intestine, although specific activity was highest in the malpighian tubules, followed by the head. The optimal pH was 8 to 9 and 1 × 10 −3 M Co 2+ and Mn 2+ ions significantly activated the enzyme, while 1 × 10 −3 M Ca 2+ and EDTA had no effect. Hg 2+ and phosphate ions inhibited it. The I 50's of some esterase inhibitors such as DEF, TOCP, DFP, K-2, OTFP, propoxur, ethyl paraoxon, n-propyl paraoxon, etc. were all greater than 1 × 10 −4 M. Purification of the enzyme from the 100,000 g supernatant of a whole body homogenate by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration resulted in a specific activity of 0.480 μmol methyl paraoxon hydrolyzed/min/mg protein with 267-fold purification. Methyl paraoxon hydrolysis was attributed to a major PAGE band as determined with 1-naphthyl acetate. The molecular weight was estimated as 120 kDa by Sephadex G-200 chromatography. The difference in the relative toxicological half-life value, ( t 0.5) e, for methyl paraoxon and ethyl paraoxon corresponded to the difference in resistance factors for these compounds in vivo. The results obtained indicate that the methyl paraoxon hydrolase of H. virescens is a true phosphorotriester hydrolase responsible in part for methyl parathion resistance in this strain of insect.