棉铃虫乙酰胆碱酯酶(AChE)的体躯分布及部分纯化

该文研究了棉铃虫Helicoverpa armigera乙酰胆碱酯酶(AChE)的体躯分布及部分纯化技术。在幼虫和成虫体内均以头部AChE活性最高;AChE在成虫和幼虫头、胸、腹部的活性分布分别为78.2%、3.9%、17.9%和90.3%、6.5%和3.2%。通过用Sepharose 4B和Sephadex G-200纯化后,AChE的最高纯化倍数为3.66和17.74倍。样品和洗脱液中含有Triton X-100时,纯化倍数明显高于无Triton X-100的处理组。     

高温对田间小菜蛾DNA损伤及4种抗药性相关酶系活性的抑制

研究了高温对福州上街菜田小菜蛾成虫4种抗药性相关酶系活性的影响。与饲养在25℃下的小菜蛾相比,33．5~C或40℃处理72h导致小菜蛾基因组DNA出现DNA凋亡特征梯度化条带。33~C饲养小菜蛾4、8、12或24h对小菜蛾成虫乙酰胆碱酯酶（AChE）和羧酸酯酶（CarE）活性无显著影响,但33℃饲养小菜蛾12或24h导致小菜蛾成虫谷胱甘肽s转移酶（GSTs）酶活性和细胞色素P450含量显著下降。36℃、24h可导致AChE活性显著下降,36℃、12h和24h可导致CarE活性显著下降,36℃、4h,8h,12h和24h可导致GST活性和细胞色素P450含量显著下降。总体上,高温对CarE、GSTs和细胞色素P450的抑制作用大于对AChE的影响,此外,3CC对AChE、CarE、GSTs酶活性和细胞色素P450含量的抑制作用大于33℃的抑制作用。     

雷公藤总生物碱对粘虫幼虫神经系统酶活性及神经递质含量的影响

【目的】明确雷公藤Tripterygium wilfordii Hook. f.生物碱对粘虫Mythimna separata (Walker)神经系统的影响,为阐明其杀虫作用机制提供依据。【方法】采用载毒叶片法测定粘虫5龄幼虫经雷公藤总生物碱处理后体内乙酰胆碱酯酶（AChE）、Na⁺, K⁺-ATPase、Ca²⁺, Mg²⁺-ATPase、谷丙转氨酶（GPT）和谷氨酸脱羧酶（GAD）等重要神经系统酶活性及乙酰胆碱（ACh）、谷氨酸（Glu）和γ-氨基丁酸（GABA）等神经递质的含量。【结果】雷公藤总生物碱处理对粘虫5龄幼虫AChE无明显影响,麻醉期处理粘虫幼虫体内ACh相对含量与同期对照无显著差异。处理粘虫幼虫在轻度麻醉期、深度麻醉期和复苏期体内GABA和Glu含量显著升高,GABA含量分别升高了89.86%, 49.28%和20.29%,Glu含量分别升高了24.55%, 23.33%和8.13%。处理粘虫幼虫GPT活性明显受到抑制,而GAD活性无明显变化。处理明显抑制粘虫幼虫头部Na⁺, K⁺-ATPase和Ca²⁺, Mg²⁺-ATPase活性,但对中肠两种ATPase活性影响不大。【结论】研究结果有助于了解雷公藤生物碱对昆虫神经系统的影响,也为进一步阐明其作用靶标奠定了基础。     

松墨天牛幼虫体内神经毒性酯酶和乙酰胆碱酯酶对甲胺磷敏感性的比较

松墨天牛Monochamus alternatus Hope是传播松材线虫Bursaphelenchus xylophilus Nickle的主要媒介昆虫,在其化学防治中经常使用有机磷杀虫剂,而这类杀虫剂有些可通过作用于神经毒性酯酶（neurotoxic esterase, NTE）使敏感的脊椎动物产生迟发性神经毒性。为了深入理解有机磷杀虫剂对害虫毒杀作用机理,增强对昆虫体内NTE活性的了解,我们在松墨天牛中开展了相关的研究。通过差异测定法和经典的乙酰胆碱酯酶（acetylcholinesterase, AChE）活性测定方法,分别测定了松墨天牛幼虫体内NTE和AChE的活性。结果发现,在松墨天牛体内存在NTE活性,其头部的NTE活力为2.80±0.30 nmol·min^-1·mg^-1,比脂肪体高出了近10倍,但仍较鸟类等脊椎动物为低。体内实验显示,松墨天牛体内NTE可被甲胺磷所抑制; 但在体外实验条件下,甲胺磷对来自松墨天牛头部的NTE活性并没有产生明显抑制作用,而此浓度下的甲胺磷对AChE表现出较高水平的抑制作用。表明松墨天牛体内NTE和AChE对甲胺磷的敏感性完全不同。     

扬子鳄纹状体AChE、SOMmRNA阳性神经元的分布

目的 观察扬子鳄纹状体内乙酰胆碱酯酶（acetylcholinesterase,AChE）和生长抑素信使核糖核酸（somatostatin messenger ribonucleic acid,SOMmRNA）阳性神经元的形态和分布.方法 采用亚铁氰化酮法和原位杂交法观察扬子鳄纹状体内AChE和SOMmRNA阳性神经元的分布和特征.结果 扬子鳄纹状体内有AChE和SOMmRNA阳性神经元分布,两种神经元均有大、中、小型细胞,以中、小型细胞为主,神经元胞体呈圆形、椭圆形、三角形、梭形和多角形.结论 扬子鳄纹状体内有AChE和SOMmRNA阳性神经元分布.     

不同发育期贝居腹盾吸虫体内乙酰胆碱酯酶分布特征

乙酰胆碱酯酶(ACh E)被证明存在于多种吸虫体内,但是关于ACh E在吸虫发育过程中的分布变化情况却少有报道。本实验以贝居腹盾吸虫(Aspidogaster conchicola)为研究对象,选取不同发育阶段的虫体,分别采用醋酸洋红染色和乙酰胆碱酯酶组织化学染色方法,对比处于不同发育阶段虫体的正常结构及乙酰胆碱酯酶分布特征,了解乙酰胆碱酯酶在吸虫发育过程中的分布变化规律。结果显示,从河蚌(Anodonta woodiana)体内获得的虫体根据内部器官的发育程度,可以分为4个不同发育阶段,即幼虫阶段Ⅰ、幼虫阶段Ⅱ、成虫阶段Ⅰ、成虫阶段Ⅱ。乙酰胆碱酯酶的分布在不同发育阶段存在着明显差异,幼虫阶段Ⅰ的睾丸及阴茎囊基部最先出现乙酰胆碱酯酶阳性反应,幼虫阶段Ⅱ在子宫末段膨大部位出现阳性反应;成虫阶段Ⅰ的卵黄腺、输卵管出现阳性反应,并且随着虫体的发育上述部位的阳性反应逐渐加强。而该吸虫的附着、消化、排泄器官在发育早期即出现较强的阳性反应,并伴随虫体发育全过程。该吸虫的神经系统在幼虫早期既已具备脑神经节、主要神经干等结构,而完整、复杂的中枢神经结构至成虫阶段才得以清晰显现。结果表明,乙酰胆碱酯酶的分布区域及反应程度随着虫体的发育而出现变化。     

土荆芥精油预处理后小菜蛾幼虫对氟虫腈敏感度的变化

采用微量点滴法测定了土荆芥Chenopodium ambrosioides精油预处理前后氟虫腈对小菜蛾Plutella xylostella 2龄幼虫的触杀作用,采用生化分析方法研究了土荆芥精油预处理前后小菜蛾幼虫体内乙酰胆碱酯酶（AChE）、羧酸酯酶（CarE）和谷胱甘肽-S-转移酶（GSTs）的比活力。研究结果表明,1μL·mL^-1和10μL·mL^-1的土荆芥精油预处理可增加小菜蛾幼虫对氟虫腈的敏感度,LC50分别为氟虫腈单用时的0.66倍和0.44倍;当用1μL·mL^-1和10μL·mL^-1土荆芥精油预处理小菜蛾2龄幼虫2h后,再用氟虫腈致死中量点滴处理,小菜蛾存活幼虫体内AChE、CarE和GSTs比活力均低于氟虫腈单独处理组。研究结果说明土荆芥精油预处理对氟虫腈的增效作用可能与主要抗性相关酶系活性变化有关。     

二化螟对杀螟硫磷产生抗性的机理

本文就二化螟Ckilo suppressalis Walker对杀螟硫磷抗性发生的机理进行了较为全面的研究。活体增效作用研究表明,脱叶磷(DEF)和氧化胡椒基丁醚(PBO)对抗性种群可分别增效1.3和34.8倍。这表明了多功能氧化酶(MFO)活性增强,而似乎与水解酶的活性无关。对寓体解毒酶的进一步研究表明,抗性和敏感种群的非特异性酯酶活力无明显差异,但羧酸酮酶(CarE,加入10^-4mol/L的毒扁豆碱)和MFO的活力在抗性种群中有所增高。R种群幼虫CarE活力分别是s的2.42和2.92倍(以a-和β-乙酸萘酯为底物);R种群幼虫MFO的O-脱甲基作用的活力为s幼虫的1.43倍(以对硝基茴香醚为底物);R幼虫乙酰胆碱酯酶(AChE)对乙酰胆碱的活力是s的1.3倍,两者相差不大。但从米氏常数K_m)和最大反应速度(V_max)看,R幼虫是s幼虫的1.9和1.6倍;以对氧磷为抑制剂的R幼虫150是s幼虫的2倍。这些表明了R和s的AChE是不同的。研究结果还表明,二化螟对杀螟硫磷抗性的机理至少包括：(1)CarE活性的增高;(2)MFO的O-脱甲基活力的增高和(3)AChE敏感性的降低等。     

二化螟对杀螟硫磷产生抗性的机理

本文就二化螟Ckilo suppressalis Walker对杀螟硫磷抗性发生的机理进行了较为全面的研究。活体增效作用研究表明,脱叶磷(DEF)和氧化胡椒基丁醚(PBO)对抗性种群可分别增效1.3和34.8倍。这表明了多功能氧化酶(MFO)活性增强,而似乎与水解酶的活性无关。对寓体解毒酶的进一步研究表明,抗性和敏感种群的非特异性酯酶活力无明显差异,但羧酸酮酶(CarE,加入10^-4mol/L的毒扁豆碱)和MFO的活力在抗性种群中有所增高。R种群幼虫CarE活力分别是s的2.42和2.92倍(以a-和β-乙酸萘酯为底物);R种群幼虫MFO的O-脱甲基作用的活力为s幼虫的1.43倍(以对硝基茴香醚为底物);R幼虫乙酰胆碱酯酶(AChE)对乙酰胆碱的活力是s的1.3倍,两者相差不大。但从米氏常数K_m)和最大反应速度(V_max)看,R幼虫是s幼虫的1.9和1.6倍;以对氧磷为抑制剂的R幼虫150是s幼虫的2倍。这些表明了R和s的AChE是不同的。研究结果还表明,二化螟对杀螟硫磷抗性的机理至少包括：(1)CarE活性的增高;(2)MFO的O-脱甲基活力的增高和(3)AChE敏感性的降低等。     

天然产物对家蚕乙酰胆碱酯酶抑制作用研究

用Sephadex G-25、DEAE-Sepharose Fast Flow和Sephadex G-200方法分离纯化家蚕头部中的乙酰胆碱酯酶(AChE),以有机磷化学农药为阳性对照,比较分析几种天然产物对AChE纯酶液与粗酶液的抑制效果.结果 表明,经过一系列步骤的纯化,最后所得AChE是纯度较高的酶液,并且纯化后的AChE对抑制作用更敏感.同时实验选取的大部分天然产物提取物对纯化后的AChE都有一定的抑制作用,其中绣线菊乙醇提取物对纯酶的IC50达到了0.091×10-3 g·mL-1.     

Tissue specific expression of the acetylcholinesterase gene in Drosophila melanogaster

Summary Acetylcholinesterase (AChE) is mainly membrane bound in the central nervous system (CNS) of larvae and in the head and thorax of adults of Drosophila melanogaster; it is mostly soluble in the larval carcass, the adult abdomen, similar to that of the embryos (Zador et al. 1986). The enzyme shows the same number of isozymes (four or five) in larvae and adults as in the head of the fly or in embryos (Zador et al. 1986). In the Df(3R)GE26/MKRS stock both the membrane bound and the soluble enzyme are at about half normal levels while in the Df(3R)Ace ^HD1/MKRS stock this is true only for the membrane bound AChE. Therefore the effect of the above deficiencies in larvae and adults is consistent with that in embryos (Zador et al. 1986). In heat-sensitive combinations of certain Ace mutant alleles both the membrane bound and the soluble enzyme has reduced activity.Abbreviations AChE acetylcholinesterase (acetylcholine acetyl hydrolase, EC 3.1.1.7) - BAP 1,5-bis(allyldimethylammonium-phenyl)-pentan-3-one dibromide - CNS central nervous system     

Two-Step Immunoaffinity Purification of Acetylcholinesterase from Rabbit Brain

Acetylcholinesterase (AChE; EC 3.1.1.7) extracted in 1% Triton X-100 from rabbit brain was purified 2,000-fold by chromatography on agarose conjugated with a monoclonal antibody directed against human red blood cell cholinesterase. After elution from the immunoadsorbent with pH 11 buffer, the preparation was purified further by affinity chromatography on phenyltrimethylammonium-Sepharose 4B with decamethonium elution. Overall yield of purified enzyme was 37% of the AChE originally solubilized, with a specific activity of 2,950 units/mg protein. Electrophoresis under reducing conditions in 7.5% sodium dodecyl sulfate polyacrylamide gels revealed only one silver-staining polypeptide band. A streamlined purification procedure enabled the isolation of electrophoretically homogeneous AChE to be completed in fewer than 7 days, at yields exceeding 50%. Electrophoretic analysis of purified AChE indicated an apparent MW of 71,000 for the monomeric subunit. Gel filtration and sucrose density gradient centrifugation in the presence of Triton X-100 showed little difference between the properties of the native and the purified enzyme. The molecular mass of the main species was estimated from the gel filtration and sedimentation data to be 280,000 daltons. Kinetic parameters of the purified protein (Km = 0.16 +/- 0.01 mM) were close to those of the native enzyme (Km = 0.12 +/- 0.01 mM) when examined with acetylthiocholine iodide as substrate. The two-step immunopurification procedure presented in this communication offers a convenient route to homogeneous neural AChE in quantities useful for detailed biochemical and immunochemical study.     

An acetylcholinesterase purified from the greenbug (Schizaphis graminum) with some unique enzymological and pharmacological characteristics

An acetylcholinesterase (AChE, EC 3.1.1.7) was purified from the greenbug, Schizaphis graminum (Rondani). The maximum velocities (Vmax) for hydrolyzing acetylthiocholine (ATC), acetyl-(beta-methyl) thiocholine (AbetaMTC), propionylthiocholine, and S-butyrylthiocholine were 78.0, 67.0, 37.4, and 2.3 micromol/min/mg, and the Michaelis constants (Km) were 57.6, 60.6, 31.3, and 33.4 microM, respectively. More than 98% of AChE activity was inhibited by 10 microM eserine or BW284C51, but only 7% of the activity was inhibited by ethopropazine at the same concentration. Based on the substrate and inhibitor specificities, the purified enzyme appeared to be a true AChE. Nondenaturing polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing of the purified AChE revealed three molecular forms. The isoelectric points were 7.3 for the major form and 6.3 and 7.1 for two minor forms. The major form of purified AChE showed molecular masses of 129 kDa for its native protein and 72 kDa for its subunits on SDS-PAGE. However, the purified AChE exhibited some distinctive characteristics including: (1) lack of affinity to the affinity ligand 3-(carboxyphenyl) ethyldimethyl ammonium, which has been used widely in purification of AChE from various insect species; and (2) 20-200-fold higher substrate-inhibition thresholds for ATC and AbetaMTC than AChE from other insect species. These biochemical properties may reflect structural differences of AChE purified from the greenbug compared with that from other insect species.     

Genetic and biochemical characterization of phosphofructokinase from Drosophila melanogaster

Phosphofructokinase (PFK;EC 2.7.1.11) activity in Drosophila melanogaster is controlled by a single dosage-sensitive region of the genome between 45F and 47E of chromosome IIR. Only a single form of PFK was detected electrophoretically in both adults and larvae. Nearly 90% of the PFK activity in adults is localized to the thorax. Purification of the enzyme was hampered by the extreme lability of Drosophila PFK; however, a 36-fold partial purification was achieved.     

Purification, characterisation, and inhibition by monoterpenes of acetylcholinesterase from the waxmoth, Galleria mellonella (L.)

Acetylcholinesterase (AChE) was purified from the brain of the waxmoth, Galleria mellonella (L.) by affinity chromatography followed by anion exchange chromatography. It resolved as a single band by polyacrylamide gel electrophoresis (PAGE) both non-denaturing and SDS (silver stained), and as a single peak by high pressure liquid chromatography (HPLC), in an overall yield of 32% representing 283-fold purification. This was a true acetylcholinesterase, with no activity as a non-specific cholinesterase (butyrylcholinesterase). The molecular weight determined by PAGE in the absence and presence of sodium dodecyl sulphate (SDS) was ca. 240,000 Da and 60,000 Da respectively, indicating an arrangement of tetrameric subunits. 2-Heptanone, the honeybee alarm pheromone, reversibly and competitively inhibited the purified Galleria AChE with a K_i value of 1.34×10⁻³ M. Furthermore, five monoterpenes associated with plant defence and representing a range of functional groups, also were reversible competitive inhibitors of the purified AChE from Galleria, which is consistent with previous data for electric eel AChE (Ryan, M.F., Byrne, O., 1988. Plant-insect coevolution and inhibition of acetylcholinesterase. Journal of Chemical Ecology 14, 1965-1975).     

Comparative studies of acetylcholinesterase purified from three field populations of Liposcelis entomophila (enderlein) (psocoptera: liposcelididae)

Acetylcholinesterace (AChE) is known to be the major target for organophophate and carbamate insecticides and biomolecular changes to AChE have been demonstrated to be an important mechanism for insecticide resistance in many insect species. In this study, AChE from three field populations of Liposcelis entomophila (Enderlein) (Psocoptera: Liposcelididae) was purified by affinity chromatography and subsequently characterized by its Michaelis‐Menten kinetics to determine if detectable changes to AChE have occurred. Bioassays revealed that the potential resistance threat of psocids in Sichuan Province (GH) was greater than either Hubei Province (WH) or Chongqing Municipality (BB). Compared to the other two populations, the WH population possessed the highest specific activity of purified AChE. Kinetic analyses indicated that the purified AChE from GH population expressed a significantly lower affinity to the substrate and a higher catalytic activity toward acetylthiocholine iodide (ATChI) (i.e., higher K_m and V_max values) than BB and WH populations. In vitro studies of AChE suggest that five inhibitors (aldicarb, eserine, BW284C51, omethoate, and propoxur) all possess strong inhibitory effects with eserine having the strongest inhibitory effect against purified AChE. According to bimolecular rate constants (k_i), the purified AChE from GH population was least sensitive to all inhibitors except for omethoate. The differences in AChE among the three populations may be partially attributed to the differences in pesticide application and control practices for psocids among the three locations. © 2010 Wiley Periodicals, Inc.     

Glutathione S-transferase isoenzymes in the two-spot ladybird, Adalia bipunctata (Coleoptera: Coccinellidae)

Isoenzymes of glutathione S-transferase (GST) in adult Adalia bipunctata, an aphidophagous predator, were studied. Cytosolic GST activity was studied in each beetle developmental stage. The highest activities towards both 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dinitro-1-iodobenzene (DNIB) occurred in adults. The enzyme distribution was investigated in adults. While most of the enzymatic activity was found in the abdomen (40-50 and 34-63% respectively) using several concentrations of both CDNB and DNIB, significant differences were observed for the head and the thorax depending on the substrate. Activities were more abundant in the thorax with DNIB (37-47%) compared to the 13-19% obtained with CDNB. Some GST activity was also detected in the elytra. GSTs were purified by epoxy-activated Sepharose 6B affinity chromatography and applied to an HPLC column to determine the native molecular weight (69 kDa). Three isoenzymes were separated by chromatofocusing at pH ranges 7-4. Three bands with molecular mass from 23 to 26 kDa were visualised on SDS-PAGE. Their isoelectric points were 6.66, 6.36, and 6.21. The substrate specificities and the kinetic parameters (Vm and Km) of the isoenzymes showed large differences depending on the isoenzyme. Arch.     

Purification and characterization of acetylcholinesterase from the Lake Van fish (Chalcalburnus tarichii Pallas, 1811)

In this study, acetylcholinesterase (AChE; EC 3.1.1.7) was purified from plasma and erythrocytes in the Lake Van fish (Chalcalburnus tarichii P.1811) by affinity chromatography. Enzymatic activity was spectrophotometrically measured according to Ellman's method, at 412 nm. Then, the optimal pH and temperature of the enzyme was determined. According to the results, the optimal pH and the optimum temperature were 8.0 and 25 degrees C, respectively. In order to control the purification of the enzyme, sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS-PAGE showed a single band for enzyme. The purification rates for plasma AChE and erythrocyte AChE are 3251.6 and 8500, respectively.     

Purification and characterization of insecticidal toxins from venom glands of the parasitic wasp, Bracon hebetor

The potency of venom from Bracon hebetor against lepidopterous larvae has been known for over 40 years, but previous attempts to purify and characterize individual protein toxins have been largely unsuccessful. Three protein toxins were purified from venom of this small parasitic wasp and the amino acid sequences of 22–31 consecutive residues at the amino-terminus were determined. These relatively large toxins (apparent molecular mass 73 kDa) were labile under many isolation techniques, but anion-exchange chromatography allowed purification with retention of biological activity. Two purified toxins were quite insecticidal (LD₅₀ < 0.3μg/g) when injected into six species of lepidopterous larvae. On a molar basis, one toxin (Brh-I) has the highest known biocidal activity against Heliothis virescens (LD₅₀ = 2 pmol/g).