脊椎动物乙酰胆碱酯酶的多糖抗原决定簇及其抑制性单克隆抗体

抗丁氏双鳍电鳐电器官乙酰胆碱酯酶（ＡＣｈＥ）的抑制性单克隆抗体ＩＨ１１（ＩｇＭ），丁氏双鳍电鳐，石纹电线电器官，比目鱼肌肉，大鼠脑，牛脑，人脑，牛红细胞及人红细胞ＡＣｂＥ均以不同的亲和层析法提纯。用酶抗原免疫测定法及酶联免疫吸附测定法检测了１Ｈ１１与上述脊椎动物的ＡＣｈＥ的交叉免疫反应性．结果表明，１Ｈ１１可与石纹电绥，比目鱼肌肉，大鼠脑、牛脑及人脑ＡＣｈＥ有交叉免疫反应。显示了１Ｈ１１所针对的抗原决定簇在进化中的高度保守性。１Ｈ１１与人，牛红细胞ＡＣｈＥ及人丁酰胆碱酯酶（ＢＣｈＥ）无交叉免疫反应性。提示哺乳类脑ＡＣｈＥ与红细胞ＡＣｈＥ及人ＢＣｈＥ有不同的抗原决定簇。１Ｈ１１对电鳐及电鳗ＡＣｈＥ活性产生明显抑制。但对其它几个脊椎动物的ＡＣｈＥ活性无抑制．表明１Ｈ１１所针对的抗原决定簇在电鳐ＡＣｈＥ的三级结构中靠近其活性中心，而在其它脊椎动物的ＡＣｈＥ中刚远离其活性中心．ＡＣｈＥ阴离子部位抑制剂依酚氟铵不影响抗原抗体反应，表明１Ｈ１１不结合在阴离子部位。Ｎ－糖苷酶Ｆ处理使ＡＣｂＥ去糖基化后。１Ｈ１１不再能与ＡＣｈＥ结合，表明至少１Ｈ１１所识别的抗原决定簇的主要部分是Ｎ－多醣．抑制性单克隆抗体１Ｈ１１对脑ＡＣｈ?     

抗电鳐乙酰胆碱酯酶单克隆抗体2E6的交叉免疫反应性^1

用乙酰胆碱酯酶（ＡＣｈＥ）偶联的ＡＨ－Ｓｅｐｈａｒｏｓｅ４Ｂ免疫亲和层析法提纯了抗丁氏双鳍电鳐ＡＣｈＥ单克隆抗体２Ｅ６（ＩｇＧ１）。用配基亲和层析法纯化了人脑，人红细胞，牛脑，牛红细胞，比目鱼肌肉及石纹电鳐电器官的ＡＣｈＥ。用酶抗原免疫测定法及固相免疫测定法观察了２Ｅ６与这些脊椎动物ＡＣｈＥ的交叉免疫反应性。结果显示２Ｅ６与石纹电鳐，人脑，牛脑及比目鱼肌肉ＡＣｈＥ有明显的交叉免疫反应性，说明２Ｅ６     

脊椎动物乙酰胆碱酯酶的多糖抗原决定簇及其抑制性单…

抗丁低双鳍电鳐电器官乙酰胆碱酯酶（ＡＣｈＥ）的抑制性单克隆抗体１Ｈ１１（ＩｇＭ），丁氏双鳍电鳐，石纹电鳐电官，比目鱼肌肉，大鼠脑，牛脑，人脑，牛红细胞及人红细胞ＡＣｈＥ均以不同的亲和层析法提纯，用酶抗原免疫测定法及不同的亲和层析法提纯，用酶抗原免疫测定法及酶联免疫吸附测定法检测了１Ｈ１１与上述脊椎动物的ＡＣｈＥ的交叉免疫反应性，结果表明，１Ｈ１１可与石纹电鳐，比目鱼肌肉，大鼠脑，牛红细胞ＡＣｈＥ及     

抗人脑乙酰胆碱酯酶单克隆抗体

本文报道３个对人红细胞乙酰胆碱酯酶（ＡＣｈＥ）无免疫交叉反应性的抗人脑ＡＣｈＥ单克隆抗体．用纯化的人脑ＡＣｈＥ抗原多次多途径免疫ＢＡＬＢ／ｃ小鼠，取脾细胞与ＳＰ２／Ｏ－Ａｇ１４小鼠骨髓瘤细胞融合，建立了３个分泌抗人脑ＡＣｈＥ单克隆抗体（均为ＩｇＭ）的杂交瘤细胞株（Ｂ１０，Ｈ１１，Ｅ１１），小鼠腹水抗体的滴度分别为１：９６０００．１：４８０００和１：２４０００．３个抗人脑ＡＣｈＥ单克隆抗体与天然电鳐电器官ＡＣｈＥ有明显的免疫交叉反应，而与人红细胞膜ＡＣｈＥ无免疫交叉反应．３个杂交瘤细胞株经３－６个月稳定传代后冻存于液氮中，一年后复苏，细胞生长良好，分泌抗体水平未见下降．     

Anionic subsite of active center of Torpedo acetylcholinesterase constructs a part of its conformati

目的：研究电鳐乙酰胆碱酯酶（ＡＣｈＥ）结构功能关系，探讨ＡＣｈＥ负矩部位是否是其构象决定簇的一个组成部分．方法：用ＥＬＩＳＡ及酶抑制实验观察ＡＣｈＥ负矩部位探针２ＰＡＭ对ＡＣｈＥ与其ＭｃＡｂ３Ｆ３之间的免疫反应性的影响．结果：ＭｃＡｂ３Ｆ３不能与２ＰＡＭ及ＡＣｈＥ的复合物反应；２ＰＡＭ浓度依赖性地降低ＭｃＡｂ３Ｆ３对ＡＣｈＥ的抑制率；但不能解离ＡＣｈＥ与３Ｆ３构成的抗原抗体复合物．结论：电鳐ＡＣｈＥ活性中心负矩部位构成其活性中心构象抗原决定簇的一部分．     

电鳐乙酰胆碱酯酶活性中心负矩部位构成其构象决定族的一部分

目的：研究电鳐乙酰胆碱酯酶（ＡＣｈＥ）结构功能关系，探讨ＡＣｈＥ负矩部位是否是其构象决定簇的一个组成部分。方法：用ＥＬＩＳＡ及酶抑制实验观察ＡＣｈＥ负矩部位探针２－ＰＡＭ对ＡＣｈＥ与其ＭｅＡｂ ３Ｆ３之间的免疫反应性的影响。结果：ＭｃＡｂ３Ｆ３不能与２－ＰＡＭ及ＡＣｈＥ的复合物反应；２－ＰＡＭ浓度依赖性地降低ＭｃＡｂ ３Ｆ３对ＡＣｈＥ的抑制率；但不能解离ＡＣｈＥ与３Ｆ３构成的抗原抗体复合物。结论     

抗还原并烷化的AChE单克隆抗体识别的抗原决定基

电器官还原并烷化的乙酰胆碱酯酶（ＲＡ－ＡＣｈＥ）的溴化氰裂解肽或又经胃蛋白酶切割的肽与抗ＲＡ－ＡＣｈＥ单克隆抗体Ｅ９、Ｆ６及Ｆ１２仍有抗原抗体反应。经溴化氰及胰蛋白酶割的肽与３者反应消失。氧化破坏ＲＡ－ＡＣｈＥ糖侧链、溴化氰裂解的ＲＡ－ＡＣｈＥ又经酶切除糖侧链后，与３者反应无变化。Ｅ９、Ｆ６及Ｆ１２所识别的抗原决定簇是多肽型，不是多糖型。     

Anticholinesterase effects of huperzine A,E2020,and tacrine in rats

目的：比较石杉碱甲、Ｅ２０２０和他克林对大鼠胆碱酯酶的抑制作用．方法：比色法测定大鼠各脑区的ＡＣｈＥ及血清中ＢｕＣｈＥ活力．结果：三药灌胃后对大鼠脑中ＡＣｈＥ均产生剂量依赖性抑制．以石杉碱甲作用最强．他克林对ＢｕＣｈＥ的抑制显著强于Ｅ２０２０和石杉碱甲，其副反应最为明显．单次经口给药后，石杉碱甲对脑内ＡＣｈＥ的抑制作用长于Ｅ２０２０和他克林．多次给药后，对胆碱酯酶的抑制作用三药均未有耐受性产生．结论：石杉碱甲是一种高选择性ＡＣｈＥ抑制剂，具有口服活性高，作用时程长，副反应小的优点，适于临床应用．     

大黄鱼肌肉胆碱酯酶的生化特性

观察了亲和层析纯化的大黄鱼肌肉胆碱酿酶与抑制剂和底物的反应。该酶显示乙酰胆碱酪酶（ＥＣ．３．１．１．７）和丁酰胆碱酯酶（ＥＣ．３．１．１．８）的双重特征，它对毒扁豆碱十分敏感。碘化硫代乙酰胆碱和碘化硫代丁酰胆碱对该酶均有底物抑制效应，乙酰胆碱酿酶特异性抑制剂ＢＷ２８４Ｃ５１对该酶抑制作用明显，丁酰胆碱酯酶抑制剂四异丙基焦磷酰胺的抑制作用较差．     

Inhibitory effects of huperzine B on cholinesterase activity in mice

目的：测试石杉碱乙的抗胆碱酯酶作用并与他克林进行比较．方法：比色法用于测定胆碱酯酶活性．结果：石杉碱乙和他克林对ＢｕＣｈＥ和ＡＣｈＥ抑制的ＩＣ５０值的比率分别为６５８和０５４．石杉碱乙对乙酰胆碱酯酶有更强的选择性抑制作用．石杉碱乙灌胃对脑内ＡＣｈＥ的抑制作用明显强于他克林．而他克林对ＢｕＣｈＥ的抑制作用强于石杉碱乙,并有严重副反应．单次灌胃石杉碱乙在４小时内对脑内ＡＣｈＥ产生较为稳定的抑制作用．结论：与他克林比较石杉碱乙是乙酰胆碱酯酶的高选择性抑制剂,灌胃时药效高,毒性低．     

Can acetylcholinesterase serve as a target for developing more selective insecticides?

Acetylcholinesterase (AChE; EC 3.1.1.7) is a primary target of many insecticides including organophosphates (OP) and carbamates (CB). Because AChE is expressed in all invertebrate and vertebrate animals as a key enzyme of the cholinergic system, the toxicity of anticholinesterase insecticides to mammals and non-target species such as beneficial insects has been a great concern. In addition, the intensive use of OP and CB insecticides has resulted in the development of resistance in many insect pests, which has limited the use of anticholinesterase insecticides. Many aces encoding AChEs have been sequenced from a variety of vertebrates, insects and other invertebrates, and crystal structures of four AChEs have been determined in the past 20 years. Although the primary motifs and the three dimensional (3D) structures of different AChEs are similar, differences among AChEs are obvious. The catalytic properties and inhibition kinetics of AChEs from different groups of insects and mammals may be quite different, and two AChEs from a single insect may also show distinct differences. These differences may provide new opportunities for designing more selective insecticides for pest management.     

Acetylcholinesterase from snake venom as a model for its nerve and muscle counterpart.

Acetylcholinesterase (AChE) plays a key role in cholinergic transmission. At the neuromuscular junction of vertebrates, for example, it allows a fine temporal control of muscle contraction. The presence of AChE in tissues devoid of cholinergic function is also well known and raises the question of its role. In particular, AChE is abundant in the venoms of Elapid snakes, except mambas. AChE purified from snake venom consists of soluble, hydrophilic monomers. Cloning of cDNA of the AChE from Bungarus fasciatus venom showed that its C-terminal peptide is very different from those of other AChEs. The partial sequence of the Bungarus fasciatus AChE gene shows that this peptide is encoded by a new alternative exon, called S for soluble and snake. It is a short very basic peptide of 15 residues. Analysis of the venom enzyme and in vitro expression experiments showed that the last eight residues are removed in the mature protein. AChEs from snake venoms vary in their sensitivity to peripheral site inhibitors, notably to fasciculins from mamba venoms. While Ophiophagus AChE is as sensitive as Torpedo enzyme (IC50 around 10(-10) M), Naja and Heamacatus AChEs are insensitive to the toxin up to a concentration of 10(-6) M Bungarus AChE has an intermediate IC50 of 10(-8) M. Analysis of its sequence reveals two major differences in the peripheral site region, compared to Torpedo or mammalian AChEs: at position 70 it contains a methionine instead of a tyrosine, and at position 285 it contains a lysine instead of an acidic residue (glutamic or aspartic acid). Modification of these residues by site-directed mutagenesis, and enzymatic analysis of modified recombinant enzymes, confirmed that these two residues are implicated in the properties of the Bungarus AChE peripheral site. The presence of an alternative exon, which generates a soluble form of AChE in venoms, raises interesting evolutionary questions: Does it exist in snakes whose venom does not contain AChE, e.g., mambas? Did this exon pre-exist, for expression in other contexts? Snake venom AChEs offer an exceptional system for analyzing the mechanism of peripheral site inhibition, because of their wide range of activities.     

Y124 at the peripheral anionic site is important for the reactivation of nerve agent-inhibited acetylcholinesterase by H oximes

The toxicity of organophosphorus (OP) nerve agents is manifested through irreversible inhibition of acetylcholinesterase (AChE) at the cholinergic synapses, which stops nerve signal transmission, resulting in a cholinergic crisis and eventually death of the poisoned person. Oxime compounds used in nerve agent antidote regimen reactivate nerve agent-inhibited AChE and halt the development of this cholinergic crisis. Due to diversity in structures of OP nerve agents, none of the currently available oximes is able to reactivate AChE inhibited by different nerve agents. To understand the mechanism for the differential activities of oximes toward AChE inhibited by diverse nerve agents in order to aid the design of new broad-spectrum AChE reactivators, we undertook site-directed mutagenesis and molecular modeling studies. Recombinant wild-type and mutant bovine (Bo) AChEs were inhibited by two bulky side-chain nerve agents, GF and VR, and used for conducting reactivation kinetics with five oximes. A homology model for wild-type Bo AChE was built using the recently published crystal structure of human AChE and used to generate models of 2-PAM and HI-6 bound to the active-sites of GF- and VR-inhibited Bo AChEs before nucleophilic attack. Results revealed that the peripheral anionic site (PAS) of AChE as a whole plays a critical role in the reactivation of nerve agent-inhibited AChE by all 4 bis-pyridinium oximes examined, but not by the mono-pyridinium oxime 2-PAM. Of all the residues at the PAS, Y124 appears to be critical for the enhanced reactivation potency of H oximes.     

乙酰胆碱酯酶抑制剂微量筛选模型的比较研究

目的为新药发现阶段乙酰胆碱酯酶(acetylcholinest-erase,AChE)抑制剂的筛选提供实用的微量筛选方法。方法分别以电鳗AChE、大鼠大脑匀浆、蛇毒AChE为酶源,以96孔板为载体,在生理pH值和温度的条件下,用正交试验法研究酶浓度、底物浓度、反应时间、显色剂浓度对酶反应速率的影响,确定酶反应最佳条件。在研究样品溶媒DMSO对酶反应的影响和SDS终止酶反应的效果后,最终确定筛选模型运行条件并对492种贵州民族药植物提取物进行筛选研究。结果采用上述3种酶源均成功构建了微量化的AChE抑制剂筛选模型。筛选结果表明,电鳗AChE有较高的筛选灵敏度,而蛇毒AChE的筛选结果则与大鼠大脑匀浆的筛选结果有较好的一致性。结论以上述3种具有代表性的酶源构建的AChE抑制剂微量筛选模型均具备了简便、快速、可靠、经济、灵活的优点。其中电鳗AChE最适合用于新药发现阶段粗提物样品的筛选,而高纯度的蛇毒AChE更适合用于从化合物库中大规模筛选AChE抑制剂。     

Quaternary salts of 2-[(hydroxyimino)methyl]imidazole. 2. Preparation and in vitro and in vivo evaluation of 1-(alkoxymethyl)-2-[(hydroxyimino)methyl]-3-methylimida zolium halides for reactivation of organophosphorus-inhibited acetylcholinesterases

A series of structurally related mono- and bis-1,3-disubstituted 2-[(hydroxyimino)methyl]imidazolium halides were evaluated in vitro for their ability to reactivate electric eel, bovine, and human erythrocyte (RBC) acetylcholinesterases (AChE) inhibited by ethyl p-nitrophenyl methylphosphonate (EPMP) and 3,3-dimethyl-2-butyl methyl-phosphonofluoridate (soman, GD). All new compounds were characterized for (hydroxyimino)methyl acid dissociation constant, nucleophilicity, octanol-buffer partition coefficient, reversible AChE inhibition, and kinetics of reactivation of EPMP-inhibited AChEs. For GD-inhibited AChEs, maximal reactivation was used to compare compounds since rapid phosphonyl enzyme dealkylation "aging" complicated interpretation of kinetic constants. For comparison, we also evaluated three known pyridinium therapeutics, 2-PAM, HI-6, and toxogonin. In vivo evaluation in mice revealed that when selected imidazolium compounds were coadministered with atropine sulfate, they were effective in providing lifesaving protection against both GD and EPMP challenges. This was a major accomplishment in the search for effective anticholinesterase therapeutics--the synthesis and preliminary evaluation of the first new monoquaternary soman antidotes with potencies superior to 2-PAM. Significantly, there was an apparent inverse relationship between in vitro and in vivo results; the most potent in vivo compounds proved to be the poorest in vitro reactivators. These results suggested that an alternative and possibly novel antidotal mechanism of protective action may be applicable for the imidazolium aldoximes. Selected compounds were also evaluated for their inhibition of AChE phosphorylation by GD and antimuscarinic and antinicotinic receptor blocking effects.     

Kinetic evidence for different mechanisms of acetylcholinesterase inhibition by (1R)- and (1S)-stereoisomers of isomalathion

Inhibition of acetylcholinesterase (AChE) by isomalathion has been assumed to proceed by expulsion of diethyl thiosuccinyl to produce O, S-dimethyl phosphorylated AChE. If this assumption is correct, AChE inhibited by (1R)- or (1S)-isomalathions should reactivate at the same rate as AChE inhibited by configurationally equivalent (S)- or (R)-isoparathion methyl, respectively, which are expected to inhibit AChE by loss of 4-nitrophenoxyl to yield O,S-dimethyl phosphorylated AChEs. Previous work has shown that rat brain AChE inhibited by (1R)-isomalathions reactivates at the same rate as the enzyme inhibited by (S)-isoparathion methyl. However, although rat brain AChE inhibited by (R)-isoparathion methyl reactivates at a measurable rate, the enzyme inhibited by (1S)-isomalathions is intractable to reactivation. This surprising finding suggests the hypothesis that (1R)- and (1S)-stereoisomers of isomalathion inhibit AChE by different mechanisms, yielding enzymatic species distinguishable by their postinhibitory kinetics. The present study was carried out to test this hypothesis by comparing kinetic constants of reactivation (k+3) and aging (k+4) of hen brain AChE and bovine erythrocyte AChE inhibited by the four stereoisomers of isomalathion and the two stereoisomers of isoparathion methyl. Both AChEs inhibited by either (1R,3R)- or (1R,3S)-isomalathion had comparable corresponding k+3 values (spontaneous and oxime-mediated) to those of AChEs inhibited with (S)-isoparathion methyl. However, spontaneous and oxime-mediated k+3 values comparable to those of (R)-isoparathion methyl could not be obtained for AChEs inhibited by (1S,3R)- and (1S,3S)-isomalathion. Comparison of k+4 values for hen brain AChE inhibited by each stereoisomer of isomalathion and isoparathion methyl corroborated that only the (1S)-isomalathions failed to produce the expected O,S-dimethyl phosphoryl-conjugated enzymes. The results for (1R)-isomalathions suggest that the mechanism of inhibition of AChE by these isomers is the expected one involving diethyl thiosuccinyl as the primary leaving group. In contrast, the results for (1S)-isomalathions are consistent with an alternative mechanism of inhibition by these isomers implicating loss of thiomethyl as the primary leaving group.     

Oxime-mediated in vitro reactivation kinetic analysis of organophosphates-inhibited human and electric eel acetylcholinesterase

Organophosphate (OP)-based pesticides and nerve agents are highly toxic compounds which interrupt the catalytic mechanism of acetylcholinesterase (AChE) by phosphorylating the hydroxyl moiety of serine residue. The inhibited enzyme can be reactivated by the nucleophilic action of oxime reactivators. To analyze the effect of different AChE sources on reactivation efficacy of reactivators, several in vivo studies have carried out using variety of AChE sources like pig, rat and monkey. Investigations on species differences provide a better insight for the development of new reactivators. Hence, present study was mainly targeted on comparative analysis of the reactivation of electric eel and human AChE inhibited by different OP. A series of butene-linked bis-pyridinium mono oximes which vary in functional groups present at the second pyridinium ring have been examined against sarin, VX, tabun and ethyl-paraoxon-poisoned AChE. In case of tabun-inhibited AChEs, tested oximes were better than reference oximes. For VX-poisoned human AChE, reactivator K251 (k_r2;1.51?mM?^??^1?min?^??¹) showed good reactivation efficacy with standard oximes. Studies stipulated that butene-linked oximes consisting of different functional moieties are good reactivators and found to have better efficacy to reactivate nerve agent-inhibited human AChE in comparison to eel AChE.     

Nonquaternary cholinesterase reactivators. 4. Dialkylaminoalkyl thioesters of alpha-keto thiohydroximic acids as reactivators of ethyl methylphosphonyl- and 1,2,2-trimethylpropyl methylphosphonyl-acetylcholinesterase in vitro

In the search for improved lipophilic centrally active acetylcholinesterase (AChE) antidotes, a series of alpha-keto thiohydroximates were prepared and evaluated for their ability to reactivate AChEs inhibited by ethyl p-nitrophenyl methylphosphonate (EPMP) and soman (GD). The compounds conformed to the general structure 4-RC6H5C-(O)C(NOH)S(CH2)nN+R'R'.X- where R = H, CH3, F, Br, Cl, OCH3, CN;R' = CH3, C2H5, i-C3H7; R' = H, CH3; X = Cl, I; and n = 2, 3. In this series, varying R substituents on the aryl ring produced compounds with oxime pKa values from 6.8 to 8.0, optimum for an AChE reactivator. Increasing lipophilicity of the amine segment correlated with reactivator potency, as did electron-withdrawing groups on the aryl moiety, presumably due to increased binding to hydrophobic sites surrounding the AChE active site. The in vitro reactivation potency of the alpha-keto thiohydroximates approaches and even surpasses that of 2-PAM and toxogonin for GD-inhibited AChE. These initial findings point to additional structure-activity relationships to assist in the design of improved antidotal compounds.     

Two possible orientations of the HI-6 molecule in the reactivation of organophosphate-inhibited acetylcholinesterase

The inhibition of acetylcholinesterase (AChE) by organophosphorus compounds (OPs) causes acute toxicity or death of the intoxicated individual. One group of these compounds, the OP nerve agents, pose an increasing threat in the world due to their possible use in the battlefield or terrorist acts. Antidotes containing oxime compounds to reactivate the inhibited enzyme are highly valued for treatment against OP poisoning. One of these reactivators, HI-6, was shown to be significantly more effective in treating soman toxicity than other oximes, such as 2-PAM, TMB4, and obidoxime. However, HI-6 was less effective in reactivating AChE inhibited by the OP pesticide, paraoxon. In this study, the mechanism for HI-6-induced reactivation of OP-AChE conjugates was investigated using mouse mutant AChEs inhibited with different OPs including organophosphate paraoxon, and several methylphosphonates. Results indicate that the HI-6 molecule may assume two different orientations in the reactivation of AChE inhibited by organophosphate and Sp methylphosphonates. These conclusions were further corroborated by reactivation studies using an analog of HI-6 in which the bispyridinium moieties are linked by a methylene bridge rather than an ether oxygen.