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昆虫乙酰胆碱酯酶基因研究进展 总被引:3,自引:0,他引:3
对昆虫乙酰胆碱酯酶(acetylcholinesterase, AChE,EC 3.1.1.7)的基因结构和表达等方面的研究进展进行了综述。分析了昆虫乙酰胆碱酯酶基因的结构,包括10个外显子的特征。对已经报道的昆虫AChE基因进行了系统归纳,并基于已知全序列的昆虫AChE基因,进行了昆虫AChE基因的分子进化分析。对昆虫AChE基因的结构特点及其功能,以及昆虫AChE基因的活性位点、AChE的变构与昆虫抗药性的关系进行了探讨。最后对昆虫AChE基因研究中存在的问题和前景进行了分析和展望。 相似文献
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乙酰胆碱酯酶参与细胞凋亡的研究进展 总被引:1,自引:0,他引:1
乙酰胆碱酯酶(Acetylcholinesterase,AChE)是主要存在于神经系统的一种水解酶,其经典功能是水解神经递质乙酞胆碱从而终止神经冲动的传递。但是近年来,研究者发现许多证据表明它具有许多“非经典”的新功能,引起了人们的广泛关注。 相似文献
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采用联合亲和层析法从人小脑及红细胞膜中纯化了AChE,纯化的人脑及红细胞AChE在SDS-PAGE上呈一主带,分子量约为66000。人脑AChE制备酯酶与酰胺酶比活性分别为1299与143U/mg,人红细胞AChE制备分别为4584与747?mg。人脑及红细胞AChE制备的酯酶与酰胺酶活性最适pH较接近,在pH7.5=8.0之间,酯酶活性底有抑制作用。IC5010.2×10^-3及3×10^-3m 相似文献
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抗性品系棉蚜乙酰胆碱酯酶和羧酸酯酶的变异 总被引:10,自引:4,他引:10
用浸叶法测定了采自我国不同地区(泰安、莱阳、南京、北京和安阳)的棉蚜品系Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ对久效磷、甲胺磷、抗蚜威和灭多威等杀虫剂的抗性水平,各棉蚜品系对杀虫剂的抗性依次为Ⅴ>Ⅳ>Ⅲ,Ⅱ>Ⅰ。进一步研究表明,Ⅴ和Ⅳ品系棉蚜乙酰胆碱酯酶对杀虫剂的敏感性显著下降,Ⅱ品系次之,Ⅲ和Ⅰ品系接近于敏感品系。Ⅴ和Ⅳ品系乙酰胆碱酯酶的Km值显著下降,表明酶发生了质的变化。不同棉蚜抗性品系的酯酶(全酯酶和羧酸酯酶)活性均显著升高,其中Ⅲ品系的酯酶活力为Ⅱ品系的2倍。Ⅴ品系羧酸酯酶Km值达2460.4 μmol/L,而Ⅳ品系仅为84.4 μmol/L,该两个品系羧酸酯酶发生了质的变化。研究结果表明,不同抗性程度的棉蚜品系均存在代谢抗性和靶标抗性。低抗水平的棉蚜品系,以代谢抗性为主,靶标抗性为辅;中抗水平的棉蚜品系,抑或由于解毒代谢酶的活性显著增强,也可能由于靶标的敏感性显著下降;而高抗水平的棉蚜品系,依赖于代谢抗性和靶标抗性的联合作用。 相似文献
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无脊椎动物乙酰胆碱酯酶研究进展 总被引:1,自引:0,他引:1
乙酰胆碱酯酶(AChE)是生物体中一种十分重要的神经递质水解酶,也是有机磷和氨基甲酸酯类杀虫剂的作用靶标。AChE在不同生物中的性质显著不同,如编码基因个数、序列保守性、表达分布及生理功能等。作为杀虫剂的主要作用靶标之一,AChE不但可以通过单个点突变引起昆虫抗药性,还能够通过多个点突变联合作用、靶标表达量变化及基因复制等方式引起抗药性并且改变昆虫的适合度代价。本文主要从AChE的基因类型、分子进化、蛋白结构、生理功能、与昆虫的抗药性关系、同一物种中不同AChE的性质等6个方面对昆虫纲、蛛形纲和线虫等无脊椎动物AChE的研究进展作一综述。 相似文献
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Expression and reconstitution of biologically active human acetylcholinesterase fromEscherichia coli
Meir Fischer Avner Ittah Ilana Liefer Marian Gorecki 《Cellular and molecular neurobiology》1993,13(1):25-38
1. | Authentic human acetylcholinesterase (AChE) was expressed inEscherichia coli under regulation of the constitutivedeo promoter or the thermoinduciblePL promoter. |
2. | To facilitate expression in the prokaryotic system, recombinant human AChE (rhAChE) cDNA was modified at the N terminus by oligonucleotide substitutions in order to replace some of the GC-rich regions by AT. These modifications did not alter the amino acid sequence but resulted in ample production of the protein. |
3. | rhAChE accumulated in the cells and reached a level of 10% of total bacterial proteins. A partially purified inactive recombinant protein was recovered from inclusion bodies. |
4. | Active rhAChE was obtained after solubilization, folding, and oxidation, although the recovery of the active enzyme was low. A 20- to 40-fold increase in enzymatically active rhAChE was achieved by replacing Cys580 by serine. |
5. | The recombinant enzyme analogue was indistinguishable from native AChE isolated from erythrocytes in terms of substrate specificity and inhibitor selectivity. |
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Immunosensors for detection of pesticide residues 总被引:2,自引:0,他引:2
Immunosensors are biosensors that use antibodies or antigens as the specific sensing element and provide concentration-dependent signals. There is great potential in the applications of immunosensing technologies for rapid detection of pesticide residues in food and environment. This paper presents an overview of various transduction systems, such as electrochemical, optical, piezoelectric, and nanomechanics methods, which have been reported in the literature in the design and fabrication of immunosensors for pesticide detection. Various immobilization protocols used for formation of a biorecognition interface are also discussed. In addition, techniques of regeneration, signal amplification, miniaturization, and antibodies are evaluated for the development and applications of these immunosensors. It can be concluded that despite some limitations of the immunosensing technologies, these immuosensors for pesticide monitoring are becoming more and more relevant in environmental and food analysis. 相似文献
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Production of recombinant human acetylcholinesterase (AChE) by a high producer human embryonic kidney cell line (293) was evaluated by three main cell propagation systems; surface propagator, fixed-bed reactor and stirred microcarrier cultures. The recombinant cell line expresses AChE levels as high as 10–20 mg/l/day. System productivities in either the surface propagator (multitray system), or in the fixed-bed reactor (polyurethane macroporous sponges) were 4–8 mg AChE/l/day during a production period of 8 days. Similar productive rates, yet longer production periods (up to 22 days), were obtained in microcarrier (MC) cultures using either polystyrene beads (Biosilon); collagen-coated dextran beads (Cytodex-3); or gelatin macroporous beads (Cultispher-G). Best results were obtained in an aggregate cculture using cellulose beads charged with diethylaminoethyl (DEAE) groups, (Servacel), as carriers. In this culture, a system productivity of 6–10 mg/l/day was maintained for 28 days. 相似文献
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Zeynep Kksal Zuhal Alm Songül Bayrak lhami Gülin Hasan
zdemir 《Journal of biochemical and molecular toxicology》2019,33(5)
Human carbonic anhydrase I and II isoenzymes (hCA I and II) and acetylcholinesterase (AChE) are important metabolic enzymes that are closely associated with various physiological and pathological processes. In this study, we investigated the inhibition effects of some sulfonamides on hCA I, hCA II, and AChE enzymes. Both hCA isoenzymes were purified by Sepharose‐4B‐L‐Tyrosine‐5‐amino‐2‐methylbenzenesulfonamide affinity column chromatography with 1393.44 and 1223.09‐folds, respectively. Also, some inhibition parameters including IC50 and Ki values were determined. Sulfonamide compounds showed IC 50 values of in the range of 55.14 to 562.62 nM against hCA I, 55.99 to 261.96 nM against hCA II, and 98.65 to 283.31 nM against AChE. Ki values were in the range of 23.40 ± 9.10 to 365.35 ± 24.42 nM against hCA I, 45.87 ± 5.04 to 230.08 ± 92.23 nM against hCA II, and 16.00 ± 45.53 to 157.00 ± 4.02 nM against AChE. As a result, sulfonamides had potent inhibition effects on these enzymes. Therefore, we believe that these results may contribute to the development of new drugs particularly in the treatment of some disorders. 相似文献
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AbstractMany pesticides applied in cut flowers can be readily absorbed through the skin of florists during preparing bouquets and handling contaminated flowers. A study was conducted among volunteer Belgian florists in order to assess their total exposure by measuring concentrations of pesticides (parent compounds and metabolites) in their urines. A total of 42 urine samples (24-h urines) were collected from florists during their professional activities, on the three most important commercial periods. The concentrations of pesticide residues and metabolites in urine samples were analyzed with a multiresidue liquid chromatography tandem mass spectrometry method, after an ethyl acetate extraction. The results are compared with those of a control group of 42 subjects not occupationally exposed to pesticides, collected in the same periods. A total of 70 residues (56 pesticides and 14 metabolites) were identified, with an average of about eight pesticide residues and metabolites per florist’s urine sample and an average total concentration per sample of 4.3 µg/g creatinine, ranging from 0.2 to 67 µg/g creatinine. Significantly higher urinary excretion of metabolites (t-test) was found in florists than in control group. These results demonstrate that Belgian florists are exposed daily to pesticide residues with a potential effect on their health. 相似文献
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Jian-Rong Gao J. Venkateswara Rao Gerald E. Wilde Kun Yan Zhu 《Archives of insect biochemistry and physiology》1998,39(3):118-125
Acetylcholinesterase (AchE, EC 3.1.1.7) was purified from western corn rootworm (WCR, Diabrotica virgifera virgifera) beetles by affinity chromatography. The purification factor reached over 20,000-fold with a specific activity of 169.5 μmol/min/mg and a yield of 23%. The Vmax values for hydrolyzing acetylthiocholine (ATC), acetyl-(β-methyl)thiocholine (AβMTC), propionylthiocholine (PTC), and S-butyrylthiocholine (BTC) were 184.8, 140.5, 150.2, and 18.8 μmol/min/mg, respectively, and Km values were 19.7, 18.5, 14.1, and 11.0 μM, respectively. The first three substrates showed significant inhibition to the AchE at higher concentrations, whereas BTC showed inhibition at the concentrations of 0.25–2 mM but activation at >4 mM. AchE activity was almost completely inhibited by 1 μM eserine and BW284C15, respectively, but only 12% of AchE activity were inhibited by ethopropazine at the same concentration. These results suggested that the purified AchE from WCR was a typical insect AchE. Insecticides or their oxidative metabolites, chlorpyrifos-methyl oxon, carbofuran, carbaryl, malaoxon, and paraoxon, used in in vitro kinetic study exhibited high inhibition to AchE purified from WCR. However, chlorpyrifos-methyl oxon and carbofuran showed at least 36- and 4-fold, respectively, higher inhibitory potency than the remaining insecticides examined. Results from our in vitro inhibition of AchE agreed quite well with the previously published in vivo bioassay data. Arch. Insect Biochem. Physiol. 39:118–125, 1998. © 1998 Wiley-Liss, Inc. 相似文献
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Guo-Jun Lang Jin-Yan Shang Yan-Xia Chen Ying-Jun Cui Qiang Wang Zhen-Hua Tang Chuan-Xi Zhang 《World journal of microbiology & biotechnology》2010,26(10):1795-1801
Acetylcholinesterase is a key enzyme of the animal nerve system. The enzyme is the primary target of organophosphorous (OP)
and carbamate (CB) insecticides. The insect AChE is being extensively used in development of new insecticides or in vitro
selection of the new designed insecticides, and in pharmacological and toxicological field. Rapid assays using AChE-based
methods have been proposed as an efficient and rapid method for the detection of pesticides, especially in many Asian markets.
In this study, the acetylcholinesterase gene was cloned from housefly (Musca domestica) susceptible to organophosphate (OP) and carbamate (CB) insecticides, and expressed in baculovirus-insect cells system using
a bioreactor with oxygen supplementation. The recombinant housefly AChE was purified using ammonium sulfate precipitation
and procainamide affinity chromatography, and approximately 0.42 mg of the purified AChE with high biological activity (118.9 U/mg)
was obtained from 100 ml of culture solution. The purified AChE was highly sensitive to OP and CBs insecticides. In conclusion,
an efficient expression and purification system has been developed for large-scale production of recombinant housefly AChE.
The recombinant enzyme is potential to be used for the detection of pesticide residues. 相似文献
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Colletier JP Fournier D Greenblatt HM Stojan J Sussman JL Zaccai G Silman I Weik M 《The EMBO journal》2006,25(12):2746-2756
Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge. 相似文献
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Adnan Cetin Fikret Türkan Parham Taslimi lhami Gulin 《Journal of biochemical and molecular toxicology》2019,33(3)
Novel substituted thiophene derivatives ( 1, 2a‐e, 3, and 4 ) were synthesized and their structures were characterized by infrared radiation, nuclear magnetic resonance, and mass analysis. These novel substituted thiophene derivatives were effective inhibitor compounds of the carbonic anhydrase I and II isozymes (hCA I and II), and acetylcholinesterase (AChE) enzyme with K i values in the range of 447.28 to 1004.65 nM for hCA I, 309.44 to 935.93 nM for hCA II, and 0.28 to 4.01 nM for AChE, respectively. Novel substituted thiophene derivatives can be good candidate drugs for the treatment of some diseases like neurological disorders, epilepsy, glaucoma, gastric and duodenal ulcers, mountain sickness, or osteoporosis as carbonic anhydrase isozymes inhibitors, and for the treatment of Alzheimer’s and Parkinson’s diseases as acetylcholinesterase inhibitors. 相似文献