Purification and characterization of acetylcholinesterase of brown planthopper, Nilaparvata lugens (stal)

• Main Authors: Liu Ya Hui, 劉雅慧
• Other Authors: Sun Chih Ning
• Format: Others
• Language: zh-TW
• Published: 1993
• Online Access: http://ndltd.ncl.edu.tw/handle/12663393437500035035

碩士 === 國立中興大學 === 昆蟲學系 === 81 === Less than 10% of the acetylcholinesterase (AChE) activity was detected in the soluble fraction (100,000 g supernatant) when adults of the brown plant- hopper, Nilaparvata lugens (St l), were homogenized and fractionated by centrifugation. Digestion of the homo- genate with phospholipase C released some AChE from the membrances and 68% of the activity could be found in the soluble fraction. The AChE activity sequentially extracted from BPH with different buffer was 10%, 57% and 14%, respectively. Affinity chromatography using m- carboxyphenyldimethylethyl ammonium gel resulted in ca. 1,200-fold purification of AChE of his planthopper. Purified AChE appeared as a broad protein bandsodium dodacyl sulfate (SDS)-PAGE with molecular mass in the range of 70-80 KDa.Four molecular forms, i.e., amphiphilic dimer and monomer, hydrophilic dimer and monomer, of purified AChE from BPH were observed on nondena- turing PAGE with amphiphilic dimer as the major form. Phospholipase C converted amphiphilic forms into hydro- philic forms by cleaving and releasing the glycerol backbone with its two fatty chains of the glycolipid anchor at C-terminus of AChE. Mercaptoethanol, through its reduction of interchain disulfide bridge on C terminus, dissociated dimers into monomers. Sequential or simultaneous treatments with mercaptoethanol and phospholipase C transformed all AChE into hydrophilic monomer. A field (F) and an isoprocarb-selected (R- MIPC) strains of BPH had ca. 2-fold higher AChE activity than a susceptible P strain. Purified AChE from these three strains appeared the same in terms of kinetic parameters, substrate specificity and molecular forms.However, urified AChE ofisoprocarb resistant BPH was 9-fold less sensitive toward the inhibition by this carbamate insecticide han that of susceptible strain.