乙酰胆碱酯酶的构效关系研究进展

乙酰胆碱酯酶是胆碱能神经传导中的一种关键酶．对其构效关系的研究一直是生命科学的重要课题,目前主要研究手段有X线衍射晶体学、分子生物学、电子顺磁共振等。本文重点介绍应用这些方法研究乙酰胆碱酯酶构效关系所取得的新进展,包括芳香族引导、后门开放、环的纳秒机制与高效催化的关系等。     

乙酰胆碱酯酶的结构与功能研究进展

乙酰胆碱酯酶是多分子型复杂蛋白质,广泛分布于各种组织,其主要功能是催化水解神经递质乙酰胆碱,近年来发现其有非胆碱能活性。同一组织不同分子型乙酰胆碱酯酶其催化机制及K_m具有相似性,但其一级结构不同。乙酰胆碱酯酶催化效率很高,其催化机制包括靠近、定向、一般酸碱催化及电荷接力系统等过程。乙酰胆碱酯酶在粗面内质网内合成,其代谢过程受某些因素的调节,在某些病理状态下酶活性发生改变。     

乙酰胆碱酯酶性质改变与昆虫抗药性的关系

乙酰胆碱酯酶是生物神经传导中的一种关键性酶,同时又是有机磷和氨基甲酸酯杀虫剂的靶标,因此一直是人们研究的热点。就近年来昆虫乙酰胆碱酯酶(AChE)在生化和分子生物学方面的研究进展、昆虫AChE基因结构及表达的变化对动力学参数、昆虫抗药性的影响机制以及害虫与天敌AChE的比较研究进行了简要综述。     

二化螟体内乙酰胆碱酯酶的分布及纯化方法

研究了二化螟Chilo suppressalis乙酰胆碱酯酶（AChE）的体躯和亚细胞分布,并用凝胶过滤层析和2种亲和层析方法从二化螟幼虫体内分离、纯化乙酰胆碱酯酶。结果表明：二化螟幼虫乙酰胆碱酯酶的活性主要集中于头部和胸部,而成虫胸部乙酰胆碱酯酶的活性最低,显著低于头部和腹部。成虫体内AChE的活性明显高于幼虫。在亚细胞的分布上,乙酰胆碱酯酶主要位于膜上（86%）,近46%的活性存在微粒体中。在3种纯化乙酰胆碱酯酶的方法中,以3-羧基苯基-乙基二甲基铵作配体的亲和层析法纯化效果最佳,乙酰胆碱酯酶的最高纯化倍数为536.05倍,产率30.46%。     

农用乙酰胆碱酯酶抑制剂研究进展

乙酰胆碱酯酶(ACh E)在生物神经传导中起着至关重要的作用,其主要功能是迅速水解胆碱部位的神经递质乙酰胆碱(ACh),终止神经冲动的传递。有机磷和氨基甲酸酯类杀虫剂是最常见的ACh E抑制剂,虽然对灭虫起到很大的作用,但是同时也会对人、哺乳动物等非靶标生物造成危害,所以需要研制出一种高效的、针对害虫的乙酰胆碱酯酶抑制剂。综述了有机磷和氨基甲酸酯类、部分生物碱类抑制剂,以及一些选择性的新型乙酰胆碱酯酶抑制剂的研究进展,以期为安全的乙酰胆碱酯酶抑制剂的研制提供参考。     

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。     

高等植物体内的乙酰胆碱

乙酰胆碱是由乙酸和胆碱形成的酯 ,分子量为146 .2 D。由于它含有季胺氮 ,故呈强碱性。在任何 p H值时都完全离子状态 ,乙酰胆碱最稳定的 p H为 3.8～ 4 .5。X衍射研究表明 ,乙酰胆碱结晶呈平面环形构象。核磁共振研究表明乙酰胆碱在溶液中也成此构象。在生物体内乙酰胆碱、胆碱乙酰基转移酶、乙酰胆碱受体和乙酰胆碱酯酶共同组成胆碱能系统 ,乙酰胆碱是胆碱能系统的核心。1　高等植物体内的乙酰胆碱1914年 Ewins在麦角菌中发现了乙酰胆碱 ,这是首次在非动物细胞中发现乙酰胆碱的报道。随后 ,人们陆续在细菌、真菌、藻类及许多种属的高等…     

大鼠回肠肌间神经丛一氧化氮合酶和乙酰胆碱酯酶双重显示法研究

本文用一氧化氮合酶和乙酰胆碱酯酶双重显示法,对大鼠回肠肌间神经丛进行了组织化学观察,结果发现三种不同染色的神经元：（１）乙酰胆碱酯酶阳性神经元（占８２％）;（２）一氧化氮合酶阳性神经元（占１６％）;（３）一氧化氮合酶和乙酰胆碱酯酶阳性神经元（占２％）。以上结果提示,一氧化氮可以与乙酰胆碱共存于大鼠回肠肌间神经丛的少数神经元内。本文还对肠肌间神经丛内神经元的类型和一氧化氮的作用进行了讨论。     

两种体色生物型桃蚜对杀虫剂敏感性差异及其与酶活力的关系

为了明确不同体色生物型桃蚜Myzus persicae (Suizer)对药剂的敏感性差异,本文采用室内生物测定和酶活力测定法检测不同体色生物型桃蚜对杀虫剂的敏感性以及与3种解毒酶和1种靶标酶活力关系。结果表明,红色生物型的敏感性明显低于绿色生物型,其中对吡虫啉的敏感性差异最大,LC50分别为6796648和284597 mg/L,红色为绿色生物型2388倍;其次为毒死蜱,LC50分别为12295798和1936816 mg/L,红色为绿色生物型635倍;两种生物型桃蚜对阿维菌素敏感性差异最小,红、绿色生物型的LC50分别为311678 和290966 mg/L,红色为绿色生物型107倍。红色生物型体内3种解毒酶（羧酸酯酶、谷胱甘肽-S-转移酶和多功能氧化酶P450）的比活力均高于绿色生物型,红色型羧酸酯酶比活力为绿色型的31倍,谷胱甘肽-S-转移酶为41倍,多功能氧化酶P450为15倍,两种体色生物型3种解毒酶的活力差异均达显著水平。两种体色生物型体内乙酰胆碱酯酶比活力没有差异,说明乙酰胆碱酯酶比活力与敏感性关系不大。     

敏感和抗性棉铃虫对溴氰菊酯毒力反应的机理研究

通过生物测定和生物化学方法比较了棉铃虫玎Helicoverpa armigera敏感和抗性种群对溴氰菊酯毒力反应及其3种解毒酶的差异。结果表明,田间抗性种群和室内药剂汰选的抗性种群对溴氰菊酯均有较高的抗性,其抗性倍数分别达到195.8和37 375倍。水解酯酶和多功能氧化酶是导致棉铃虫对溴氰菊酯产生高抗性的重要酶系。特异性抑制剂活体内外抑制作用测试发现,敏感种群和抗性种群均含有较高量的乙酰胆碱酯酶,但两个种群对抑制剂的亲和力反应不同,表明乙酰胆碱酯酶在敏感种群和抗性种群中发生了不同的变化,这种变化可能与棉铃虫对溴氰菊酯的抗性有关。由此推断,棉铃虫对拟除虫菊酯这类中枢神经系统神经毒剂产生抗性,乙酰胆碱酯酶发生变化可能也是一个重要因子。     

Tetrameric (G4) Acetylcholinesterase: Structure, Localization, and Physiological Regulation

Abstract: Acetylcholinesterase (AChE), a highly conserved enzyme in the animal kingdom, is distributed throughout a wide range of vertebrate tissues where it is expressed as multiple molecular forms comprising different arrangements of catalytic and structural subunits. The major AChE form in the CNS is an amphiphilic globular tetramer (G₄ AChE) consisting of four identical catalytic subunits attached to cellular membranes by a hydrophobic noncatalytic subunit (P-subunit). This study focuses primarily on current data involving the structure of the G₄ AChE P-subunit, the expression and regulation of G₄ AChE during development and adulthood, and its role(s) in certain neurological disorders including Alzheimer's disease.     

Bovine brain acetylcholinesterase primary sequence involved in intersubunit disulfide linkages

Three distinct classes of membrane-bound acetylcholinesterases (AChEs) have been identified. A12 AChE is composed of 12 catalytic subunits that are linked to noncatalytic collagen-like subunits through intersubunit disulfide bonds. G2 AChE is localized in membranes by a glycoinositol phospholipid covalently linked to the C-terminal amino acid. Brain G4 AChE involves two catalytic subunits linked by a direct intersubunit disulfide bond while the other two are disulfide-linked to a membrane-binding 20-kDa noncatalytic subunit. Molecular cloning studies have so far failed to find evidence of more than one AChE gene in any organism although alternative splicing of torpedo AChE mRNA results in different C-terminal sequences for the A12 and G2 AChE forms. Support for a single bovine AChE gene is provided in this report by amino acid sequencing of the N-terminal domains from the G2 erythrocyte, G4 fetal serum, and G4 brain AChE. Comparison of the 38-amino acid sequences reveals virtually complete identity among the three AChE forms. Additional extensive identity between the fetal serum and brain AChEs was demonstrated by sequencing several brain AChE peptides isolated by high performance liquid chromatography after trypsin digestion of nitrocellulose blots of brain AChE catalytic subunits. Cysteines involved in intersubunit disulfide linkages in brain AChE were reduced selectively with dithiothreitol in the absence of denaturants and radioalkylated with iodoacetamide. The observed sequence of the major radiolabeled tryptic peptide was C*SDL, where C* was the radioalkylated cysteine residue. This sequence is precisely the same as that observed at the C terminus of fetal bovine serum AChE and shows close homology to the C-terminal sequence of torpedo A12 AChE. We conclude that the mammalian brain G4 AChEs utilize the same exon splicing pattern as the A12 AChEs and that factors other than the primary sequence of the AChE catalytic subunits dictate assembly with either the collagen-like or the 20-kDa noncatalytic subunits.     

Structure of heparin-derived tetrasaccharides.

Quantitative solubilization of the phospholipid-associated form of acetylcholinesterase (AChE) from Torpedo electric organ can be achieved in the absence of detergent by treatment with phosphatidylinositol-specific phospholipase C (PIPLC) from Staphylococcus aureus [Futerman, Low & Silman (1983) Neurosci. Lett. 40, 85-89]. The sedimentation coefficient on sucrose gradients of AChE solubilized in detergents (DSAChE) varies with the detergent employed. However, the coefficient of AChE directly solubilized by PIPLC is not changed by detergents. Furthermore, PIPLC can abolish the detergent-sensitivity of the sedimentation coefficient of DSAChE purified by affinity chromatography, suggesting that one or more molecules of phosphatidylinositol (PI) are co-solubilized with DSAChE and remain attached throughout purification. DSAChE binds to phospholipid liposomes, whereas PIPLC-solubilized AChE and DSAChE treated with PIPLC do not bind even to liposomes containing PI. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows that PIPLC-solubilized AChE, like unmodified DSAChE, is a catalytic subunit dimer; electrophoresis in the presence of reducing agent reveals no detectable difference in the Mr of the catalytic subunit of unmodified DSAChE, of AChE solubilized by PIPLC and of AChE solubilized by Proteinase K. The results presented suggest that DSAChE is anchored to the plasma membrane by one or more PI molecules which are tightly attached to a short amino acid sequence at one end of the catalytic subunit polypeptide.     

Primary structure of a collagenic tail peptide of Torpedo acetylcholinesterase: co-expression with catalytic subunit induces the production of collagen-tailed forms in transfected cells.

The asymmetric forms of cholinesterases are synthesized only in differentiated muscular and neural cells of vertebrates. These complex oligomers are characterized by the presence of a collagen-like tail, associated with one, two or three tetramers of catalytic subunits. The collagenic tail is responsible for ionic interactions, explaining the insertion of these molecules in extracellular basal lamina, e.g. at neuromuscular endplates. We report the cloning of a collagenic subunit from Torpedo marmorata acetylcholinesterase (AChE). The predicted primary structure contains a putative signal peptide, a proline-rich domain, a collagenic domain, and a C-terminal domain composed of proline-rich and cysteine-rich regions. Several variants are generated by alternative splicing. Apart from the collagenic domain, the AChE tail subunit does not present any homology with previously known proteins. We show that co-expression of catalytic AChE subunits and collagenic subunits results in the production of asymmetric, collagen-tailed AChE forms in transfected COS cells. Thus, the assembly of these complex forms does not depend on a specific cellular processing, but rather on the expression of the collagenic subunits.     

Influence of the water structure on the acetylcholinesterase efficiency

We have studied the catalytic efficiency of acetylcholinesterase (AChE) in various solutions with ion-disturbed water structure to explore the role that the water structure plays in the substrate-enzyme encounter. The extent of water structuring in the different aqueous solutions was determined by near-infrared spectroscopy. The influence of water structure on the degree of solvation and on the intramolecular mobility of AChE was investigated for different aqueous ionic solutions by small-angle x-ray scattering technique and depolarization fluorescence spectroscopy. It was found that the encounter process between AChE and acetylthiocholine was promoted in solutions with less structured water. In these solutions it was also found that AChE is less solvated coinciding with higher intramolecular mobility. The found experimental results suggest that the water structure may influence the substrate-enzyme encounter process by diminishing the AChE solvation shell and may help diffusion of the substrate through the gorge by enhancing the intramolecular mobility of AChE.     

Which acetylcholinesterase functions as the main catalytic enzyme in the Class Insecta?

Most insects possess two different acetylcholinesterases (AChEs) (i.e., AChE1 and AChE2; encoded by ace1 and ace2 genes, respectively). Between the two AChEs, AChE1 has been proposed as a major catalytic enzyme based on its higher expression level and frequently observed point mutations associated with insecticide resistance. To investigate the evolutionary distribution of AChE1 and AChE2, we determined which AChE had a central catalytic function in several insect species across 18 orders. The main catalytic activity in heads was determined by native polyacrylamide gel electrophoresis in conjunction with Western blotting using AChE1- and AChE2-specific antibodies. Of the 100 insect species examined, 67 species showed higher AChE1 activity; thus, AChE1 was considered as the main catalytic enzyme. In the remaining 33 species, ranging from Palaeoptera to Hymenoptera, however, AChE2 was predominantly expressed as the main catalytic enzyme. These findings challenge the common notion that AChE1 is the only main catalytic enzyme in insects with the exception of Cyclorrhapha, and further demonstrate that the specialization of AChE2 as the main enzyme or the replacement of AChE1 function with AChE2 were rather common events, having multiple independent origins during insect evolution. It was hypothesized that the generation of multiple AChE2 isoforms by alternative splicing allowed the loss of ace1 during the process of functional replacement of AChE1 with AChE2 in Cyclorrhapha. However, the presence of AChE2 as the main catalytic enzyme in higher social Hymenoptera provides a case for the functional replacement of AChE1 with AChE2 without the loss of ace1. The current study will provide valuable insights into the evolution of AChE: which AChE has been specialized as the main catalytic enzyme and to become the main target for insecticides in different insect species.     

Acetylcholinesterase function is dispensable for sensory neurite growth but is critical for neuromuscular synapse stability

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine. In addition, AChE is thought to play several 'non-classical' roles that do not require catalytic function. Most prominent among these is facilitation of neurite growth. Here, we report that the zebrafish zieharmonika (zim) locus encodes AChE. We show that one mutant zim allele is caused by a pre-mature stop codon, resulting in a truncated protein that lacks both the catalytic site and the carboxy-terminal neuritogenic domain. To explore the 'non-classical' role of AChE, we examined embryos mutant for this allele. In contrast to previous results using a catalytic-inactive allele, our analysis demonstrates that AChE is dispensable for muscle fiber development and Rohon-Beard sensory neuron growth and survival. Moreover, we show that in the absence of AChE, acetylcholine receptor clusters at neuromuscular junctions initially assemble, but that these clusters are not maintained. Taken together, our results demonstrate that AChE is dispensable for its proposed non-classical roles in muscle fiber formation and sensory neuron development, but is crucial for regulating the stability of neuromuscular synapses.     

Generation of subunit-specific antibody probes for Torpedo acetylcholinesterase: cross-species reactivity and use in cell-free translations

The assembly of the collagen tailed A12 form of acetylcholinesterase (AChE) is regulated by muscle contraction. To begin to study this regulation, we derived antibody probes for the three subunits (100 kd, catalytic, and collagen tail) of AChE purified from Torpedo californica electric tissue. These included a polyclonal antiserum that recognizes all 3 subunits and 19 monoclonal antibodies; 16 of the monoclonals recognized the catalytic subunit, 2 recognized the tail subunit, and 1 recognized the 100 kd subunit on Western blots. We used immunohistochemical procedures to show that several of the anticatalytic and one of the antitail monoclonals cross-reacted with frog muscle AChE and Western blotting to show that several of the anticatalytic monoclonals cross-react with rat brain AChE. These antibodies were then used to immunoprecipitate AChE precursors from a cell-free translation system. There were generally three primary translation products, corresponding to the three enzyme subunits. Therefore, each subunit is probably derived from a separate mRNA. Occasionally there were two translation products corresponding to the catalytic subunit alone. The catalytic subunit was glycosylated following addition of canine microsomal membranes to the translation mix. The mRNA coding for this subunit appeared to be present in the poly(A)- RNA pool.     

Effect of the state of the lipid phase of the membrane on the effectiveness of photochemical modification of erythrocyte acetylcholinesterase

Modification of the lipid phase structure of the erythrocyte membrane by phospholipases A2, C and D as well as the partial depletion of cholesterol was shown to be accompanied by the change of the acetylcholinesterase (AChE) UV-sensitivity. The ability of UV-light to change the catalytic properties (Km) of the membrane-bound AChE not observed for free AChE (constant value of Km) and known as the phenomenon of photochemical allotopy, is retained in the cholesterol depleted membranes and disappears after an enzymatic treatment of the membranes by phospholipases. The possible non-photochemical influence of the membrane lipid phase in response to UV-damage of membrane-bound AChE is discussed.     

Monoclonal Antibodies Specific for the Different Subunits of Asymmetric Acetylcholinesterase from Chick Muscle

The asymmetric (20S) acetylcholinesterase (AChE, EC 3.1.1.7) from 1-day-old chick muscle, purified on a column on which was immobilised a monoclonal antibody (mAb) to chick brain AChE, was used to immunise mice. Eight mAbs against the muscle enzyme were hence isolated and characterised. Five antibodies (4A8, 1C1, 10B7, 7G8, and 8H11) recognise a 110-kilodalton (kDa) subunit with AChE catalytic activity, one antibody (7D11) recognises a 72-kDa subunit with pseudocholinesterase or butyrylcholinesterase (BuChE, EC 3.1.1.8) catalytic activity, and two antibodies (6B6 and 7D7) react with the 58-kDa collagenous tail unit. Those three polypeptides can be recognised together in the 20S enzyme used, which is a hybrid AChE/BuChE oligomer. Antibodies 6B6 and 7D7 are specific for asymmetric AChE. Four of the mAbs recognising the 110-kDa subunit were reactive with it in immunoblots. Sucrose density gradient analysis of the antibody-enzyme complexes showed that the anti-110-kDa subunit mAbs cross-link multiple 20S AChE molecules to form large aggregates. In contrast, there is only a 2-3S increase in the sedimentation constant with the mAbs specific for the 72-kDa or for the 58-kDa subunit, suggesting that those subunits are more inaccessible in the structure to intermolecular cross-linking. The 4A8, 10B7, 7D11, and 7D7 mAbs showed cross-reactivity to the corresponding enzyme from quail muscle; however, none of the eight mAbs reacted with either enzyme type from mammalian muscle or from Torpedo electric organ. All eight antibodies showed immunocytochemical localisation of the AChE form at the neuromuscular junctions of chicken twitch muscles.