Purification and characterization of carboxylesterases of a rice green leafhopper Nephotettix cincticeps Uhler
Purification and characterization of carboxylesterases of a rice green leafhopper Nephotettix cincticeps Uhler
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DOI：
10.1006/pest.1996.0022
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发表时间：
1996-03-01
影响因子：
4.7
通讯作者：
Sun, CN
中科院分区：
文献类型：
--
作者：
Chiang, SW;Sun, CN
采用等电聚焦电泳法，结合硫酸铵分级、凝胶过滤、层析等方法分离纯化了稻纵卷叶蝉匀浆中的4种以上的羧酸酯酶同工酶。分离得到4个组分，分别为E_1、E_2、E_3和E_4，等电点为5.1~4.85。最丰富的E3的相对分子质量为58.6 kDa，在SDS-PAGE上呈现均一的电泳带。[1，3-H-3]二异丙基氟磷酸盐标记实验表明，E_2的次要成分和E_4的主要成分--58.6 kDa的蛋白质是所寻找的羧酸酯酶同工酶。一种分子量相同的蛋白质在F1中以微量存在，经考马斯亮蓝染色在SDS-PAGE上几乎检测不到，实际上是pI 5.1的羧酸酯酶同工酶。所有四个组分对几种模型底物都表现出显著的活性，其中α-萘丁酸酯是最受欢迎的。它们对马拉硫磷、氯氰菊酯和氯氰菊酯的活性比它们对模型底物的活性低约10(6)倍。拟除虫菊酯比马拉硫磷更容易被这些水解酶降解，顺式氯氰菊酯比反式氯氰菊酯更容易被水解，E4是唯一能与褐飞虱羧酸酯酶抗血清发生交叉反应的组分。在四个同工酶组分中，含量最丰富的E3对所有四种杀虫剂的活性都低得令人惊讶，实际上对顺式氯氰菊酯的活性最低。与敏感品系相比，野生品系对模型底物的羧酸酯酶活性高26到37倍。然而，这两个菌株在马拉硫磷、氯氰菊酯和氯氰菊酯的水解率上几乎没有差异。野外菌株产生的羧酸酯酶至少是敏感菌株的8倍。(C)1996年学术出版社。Inc.
More than four carboxylesterase isozymes in the homogenate of a rice green leafhopper, Nephorettix 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-H-3]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 Fl 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 acid 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. The field strain produced at least eight times more carboxylesterases than the susceptible strain. (C) 1996 Academic Press. Inc.