Purification and characterization of carboxylesterases of a rice green leafhopper Nephotettix cincticeps Uhler

More than four carboxylesterase isozymes in the homogenate of a rice green leafhopper, Nephotettix cincticeps Uhler, could be resolved by isoelectric focusing electrophoresis. A combination of ammonium sulfate fractionation, gel filtration, and chromatofocusing chromatography was used to isolate and...

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Bibliographic Details
Published in:Pesticide biochemistry and physiology 1996-03, Vol.54 (3), p.181-189
Main Authors: Chiang, S.W. (National Chung-Hsing University, Taichung, Taiwan, Republic of China.), Sun, C.N
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
Biological and medical sciences
CARBOXILESTERASA
CARBOXYLESTERASE
Chemical control
Cicadellidae
CIPERMETRIN
Control
CYPERMETHRINE
Fundamental and applied biological sciences. Psychology
Homoptera
INSECTICIDAS
INSECTICIDE
ISOENZIMAS
ISOENZYME
MALATHION
MALATION
NEPHOTETTIX CINCTICEPS
PERMETHRINE
PERMETRINA
Phytopathology. Animal pests. Plant and forest protection
Protozoa. Invertebrates
PURIFICACION
PURIFICATION
RESISTANCE AUX PRODUITS CHIMIQUES
RESISTENCIA A PRODUCTOS QUIMICOS
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Summary:More than four carboxylesterase isozymes in the homogenate of a rice green leafhopper, Nephotettix cincticeps Uhler, 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 pI 5.1. All four fractions exhibited significant activity toward several model substrates with alpha-naphthyl butyrate being the most preferred. Their activity toward malathion, permethrin, and cypermethrin was ca. 10(6)-fold lower than their activity toward the model substrates. The pyrethroids were hydrolyzed more readily than malathion by these hydrolases, and cis-permethrin was more preferred than the trans-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 toward cis-permethrin and cypermethrin. A field strain of N. cincticeps had 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
ISSN:0048-3575
1095-9939
DOI:10.1006/pest.1996.0022