Purification and Characterization of Carboxylesterases of a Rice Green LeafhopperNephotettix cincticepsUhler

Abstract

More than four carboxylesterase isozymes in the homogenate of a rice green leafhopper,Nephotettix cincticepsUhler, could be resolved by isoelectric focusing electrophoresis. A combination of ammonium sulfate fractionation, gel filtration, and chromatofocusing chromatography was used to isolate and purify these isozymes. Four fractions, i.e., E1, E2, E3, and E4, with pI's ranging from 5.1 to 4.85, were obtained. The most abundant E3 had a molecular mass of 58.6 kDa and appeared electrophoretically homogeneous on SDS–PAGE. [1,3-3H]Diisopropyl fluorophosphate-labeling experiment revealed that the proteins of 58.6 kDa, a minor component of E2 and the major component of E4, were the carboxylesterase isozymes sought. A protein of the same molecular weight which existed in a very minute amount in E1 and was barely detectable on SDS–PAGE by Coomassie blue staining was actually the carboxylesterase isozyme of pI5.1. All four fractions exhibited significant activity toward several model substrates with α-naphthyl butyrate being the most preferred. Their activity toward malathion, permethrin, and cypermethrin was ca. 106-fold lower than their activity toward the model substrates. The pyrethroids were hydrolyzed more readily than malathion by these hydrolases, andcis-permethrin was more preferred than thetrans-isomer. E4 was the only fraction that cross-reacted with the antiserum against carboxylesterases of a rice brown planthopper,Nilaparvata lugens.Among the four isozyme fractions, E3, the most abundant, showed surprisingly low activity toward all four insecticides and was actually the least active fraction towardcis-permethrin and cypermethrin. A field strain ofN. cincticepshad 26- to 37-fold higher carboxylesterase activity toward the model substrates than a susceptible strain. Yet, little, if any, difference in the hydrolysis of malathion, permethrin, and cypermethrin was observed between these two strains. The field strain produced at least eight times more carboxylesterases than the susceptible strain.