灭多威对日本青鳉和大型溞乙酰胆碱酯酶的效应研究

[目的]研究不同浓度的灭多威对大型溞和日本青鳉乙酰胆碱酯酶(AChE)活性的影响。[方法]通过测定灭多威对大型溞和日本青鳉的24 h LC50评价其毒性大小,并采用Ellman方法检测灭多威对生物体AChE活性的影响。[结果]灭多威对大型溞幼溞和日本青鳉的24 h LC50分别为9.83和185μg/L。随着灭多威暴露浓度的增加,对大型溞幼溞和日本青鳉AChE活性的抑制作用增强。在体内试验中,在灭多威连续暴露下乙酰胆碱酯酶的相对酶活总体呈下降趋势。在体外试验中,在一定的时间范围内灭多威对AChE活性的抑制率与暴露时间呈一定的线性关系,且随着暴露时间的延长而增大。[结论]体内和体外试验的结果表现出明显的差异性,因此在研究污染物对生物体酶活的影响时,应分别进行体内和体外试验。     

滤纸壳聚糖膜固定乙酰胆碱酯酶的研究

[目的]探索滤纸壳聚糖膜固定乙酰胆碱酯酶(AChE)的最佳条件。[方法]以滤纸壳聚糖膜为载体,戊二醛为交联剂,牛血清白蛋白(BSA)为保护剂,进行AChE的固定。并对固定化酶的理化性质进行研究。[结果]固定AChE的最佳条件为将50μl 150 U/mlAChE液,50μl 5%(V/V)戊二醛溶液,100μl 1%(W/W)BSA,pH 8.0的0.2 mol/L的PBS缓冲液配制成1 ml酶液,滤纸壳聚糖膜浸于该酶液4℃固定8 h。固定化酶的最适反应温度为37℃,最适pH为8.0,能够重复利用4次以上。[结论]该研究为利用AChE快速检测蔬菜水果中有机磷农药残留提供了理论依据。     

烟粉虱与温室白粉虱羧酸酯酶、谷胱甘肽转移酶和乙酰胆碱酯酶性质的比较研究

对温室白粉虱 Trialeurodes vaporariorum和烟粉虱 Bemisia tabaci羧酸酯酶(Car E)、乙酰胆碱酯酶 (ACh E)和谷胱甘肽转移酶 (GSTs)的生物化学性质进行了初步研究。比较 Car E活性 ,烟粉虱显著高于温室白粉虱 ,烟粉虱和温室白粉虱 Car E对底物乙酸 -α-萘酯的 Km 分别为 0 .073mmol/ L 和 3.458mmol/ L。烟粉虱 ACh E活性分布高峰在 2～3m OD/ (min·头 )之间 ,而温室白粉虱 ACh E活性分布高峰在 10～ 15 m OD/ (min·头 )之间 ,烟粉虱 ACh E的 Km 值是温室白粉虱 Km 值的 12 .6倍。烟粉虱的 GSTs比活力为2 .218OD/ (mg pro·min) ,显著高于温室白粉虱的 GSTs比活力 0 .663OD/ (mg pro· min) ,差异达极显著水平 ,烟粉虱 GSTs对底物的亲和力比温室白粉虱高。     

Boric acid toxicity to the German cockroach, Blattella germanica: Alterations in midgut structure, and acetylcholinesterase and glutathione S-transferase activity

Oral toxicity of boric acid, an inorganic insecticide, was evaluated on German cockroach, Blattella germanica L. (Dictyoptera, Blattellidae). Newly emerged adults were exposed to various concentrations of boric acid incorporated into the diet. Results showed that treated insects exhibited toxic symptoms with a dose-dependent mortality. Histological study of midgut revealed alterations in the epithelial cells and a significant increase in the epithelium thickness. In a second series of experiments, the compound was investigated on the activities of acetylcholinesterase (AChE) and glutathione S-transferases (GSTs). Data showed that the compound induced GSTs and reduced the activity of AChE. From this experiment, it may be concluded that ingested boric acid caused death of insects perhaps ultimately by starvation via alterations of the midgut. Additionally, the compound seemed to present a neurotoxic action as evidenced by the symptoms of poisoning and the reduction in AchE activity.     

球孢白僵菌侵染对桑天牛幼虫解毒酶活性的影响

为明确球孢白僵菌(Beauveria bassiana)对桑天牛(Apriona germari Hope)的致病机制,探讨了球孢白僵菌侵染对桑天牛幼虫解毒酶系的影响,对用球孢白僵菌处理后不同时间桑天牛幼虫体内羧酸酯酶、谷胱甘肽-S-转移酶和乙酰胆碱酯酶活性进行了测定。结果表明球孢白僵菌对上述3种酶的活性有显著影响,其中对羧酸酯酶和谷胱甘肽-S-转移酶活性的影响,均表现出先刺激后抑制的作用;而对乙酰胆碱酯酶活性的影响则呈先抑制后激活再抑制再激活的趋势。     

乙酰胆碱酯酶的晶体结构及功能位点

乙酰胆碱酯酶晶体结构研究表明 ,ACh E存在一个深而窄的谷 ,谷底部有一个活性位点 ,它是由催化三联体和所谓的阴离子亚位点组成。阴离子亚位点识别底物的四价铵基团 ;由 Glu32 7、His4 4 0和 Ser2 0 0构成的催化三联体负责底物乙酰胆碱的水解。在谷的入口处 ,有一个调节位点被称为外周阴离子位点 ,残基 Trp84和 Trp2 79分别在催化和外周的阴离子位点起关键作用。朝着谷底部方向存在一个强的静电偶极子 ,该偶极子对阳离子产物胆碱的清除是非常不利的 ,并与酶的高催化效率形成明显对照。位于催化口袋底部的后门开放为产物释放提供了另一种途径 ,但对后门学说的争论仍然十分激烈。X-衍射晶体学与化学修饰研究有助于阐明配基结合活性中心谷特异性的分子基础。位点特异性突变分析在确定氨基酸残基参与乙酰胆碱酯酶空间结构或催化功能中起着非常重要的作用。     

萝卜叶不同萃取物对乙酰胆碱酯酶抑制活性研究

[目的]研究萝卜叶不同萃取组分对乙酰胆碱酯酶的抑制活性。[方法]通过对萝卜叶水提原液、水提不同萃取组分及萃取后的水相进行乙酰胆碱酯酶抑制活性测试,对萝卜叶抑制乙酰胆碱酯酶能力进行评价。[结果]以石杉碱甲为对照,萝卜叶不同萃取物对乙酰胆碱酯酶的抑制能力从大到小依次为石杉碱甲、二氯甲烷组分、乙酸乙酯组分、石油醚组分、正丁醇组分、水相、萝卜叶水提原液。[结论]萝卜叶具有较好的乙酰胆碱酯酶抑制活性,并呈现一定的浓度依赖性。     

宋果灵对乙酰胆碱酯酶活性的抑制作用

[目的]认识宋果灵具体的杀虫机制,为开发此类植物源农药以及进行酶法筛选提供毒理学参考。[方法]配制浓度分别为0、0.005、0.01、0.020、.0350、.05 mg/ml的宋果灵,研究宋果灵对乙酰胆碱酯酶活性的抑制作用及可能的机制。[结果]随着宋果灵浓度的升高,乙酰胆碱酯酶活性逐渐下降,说明宋果灵对乙酰胆碱酯酶活性有抑制作用,线性回归方程为:y=-0.118 9x+99.387,R2=0.934 4,IC50=0.412 mg/ml。宋果灵含有N原子,与质子结合后,带正电,NH4+正离子与Trp84、Phe330的侧链芳环形成阳离子-π相互作用,从而抑制了酶活性。[结论]宋果灵可以作为农药先导化合物,用于合成高效、低毒、无公害的新农药。     

柞蚕饰腹寄蝇不同地理种群的杀虫剂靶标酶和解毒酶活性分析

昆虫通过降低靶标酶敏感性及增强对杀虫剂的代谢能力产生抗药性。蝇毒磷是防治柞蚕饰腹寄蝇(Blepharipa tibia-lis Chao)的常用有机磷杀虫剂,为探讨柞蚕饰腹寄蝇不同地理种群对蝇毒磷的抗药性分化程度,分析来自辽宁省瓦房店市、凤城市、庄河市、开原市和西丰县的5个柞蚕饰腹寄蝇地理种群体内的乙酰胆碱酯酶(AChE)、谷胱甘肽S-转移酶(GST)、细胞色素P450(CYP450)和羧酸酯酶(CarE)4种酶的活性及蝇毒磷对这些酶活性的体外抑制率。结果表明:来自瓦房店市和凤城市的柞蚕饰腹寄蝇地理种群的AChE比活力及蝇毒磷对酶活性的体外抑制率均显著高于其它3个地理种群;瓦房店市地理种群的GST比活力显著高于其它4个地理种群,蝇毒磷对酶活性的体外抑制率显著高于除庄河市地理种群外的其它3个地理种群;庄河市地理种群的CYP450比活力显著高于来自瓦房店市、凤城市和西丰县的3个地理种群,但与开原市地理种群相比差异不显著,蝇毒磷对瓦房店市地理种群的CYP450活性的体外抑制率显著高于其它4个地理种群;瓦房店市与凤城市地理种群的CarE比活力均显著高于来自庄河市、开原市和西丰县的3个地理种群;蝇毒磷对瓦房店市和凤城市地理种群的CarE活性的体外抑制率显著高于庄河市地理种群,对其它地理种群间的CarE活性的体外抑制率差异不显著。根据以上检测结果,推测来自庄河市的柞蚕饰腹寄蝇地理种群对蝇毒磷的抗性相对较强,而来自瓦房店市的柞蚕饰腹寄蝇地理种群对蝇毒磷的抗性相对较弱。     

Comparisons of Properties of Acetylcholinesterase from Two Field-Collected Populations of Oxya chinensis Thunberg (Orthoptera: Acrididae) and the Role of Acetylcholinesterase in the Susceptibility to Malathion

In this study, acetylcholinesterase (AChE) was extracted from two field-collected populations of Oxya chinensis (Xinxiang City, Henan Province and Changzhi City, Shanxi Province). AChE activities were decreased when concentrations of ATC increased, showing a characteristic phenomenon of substrate inhibition at high concentration in both populations. Such inhibition occurred at relatively low concentration for AChE from Xinxiang population but relatively high for AChE from Changzhi population. The kinetic study showed that there were no significant differences between the two populations in the Km values. The Km value in Changzhi population was only 1.09-fold higher than that in Xinxiang population. However,significant differences were observed between the two populations in Vmax values. The Vmaxvalue in Changzhi population was 1.32-fold higher than that in Xinxiang population. The inhibition study in vitro showed that the AChE from both populations exhibited similar rank order in sensitivity to inhibition by three OPs, as determined by comparison of their bimolecular rate constants (ki), from the most potent inhibition to the least was chlopyrifos-oxon ＞ paraoxon ＞demeton-s-methyl for AChE from the two populations and that the ki values in Xinxiang population were lower than those in Changzhi population. The I50 values of AChE from Xinxiang population were 4.84-, 2.66-, and 1.92-fold less sensitive to inhibition by paraoxon, chlopyrifos-oxon, and demeton-s-methyl. These results were consistent with the results in bioassay. It is inferred that AChE insensitivity to OP insecticides plays an important role in the differences of insusceptibility of Oxya chinensis to malathion between the two populations.