有机磷水解酶的表面显示技术研究进展

有机磷化合物在农业中的广泛应用,给农作物带来增产的同时对环境造成污染,严重威胁着人类的健康,其生物解毒已受到高度重视.有机磷水解酶(OPH)是目前处理有机磷化合物最有效的水解酶类,而通过基因工程手段获得高表达、高降解效率的OPH,尤其是OPH的表面显示技术是近年来的研究热点.主要综述了大肠杆菌、假单胞菌、酿酒酵母的OPH表面显示技术研究及应用进展和发展趋势.     

建立更好的抗毒素

丁酰胆碱酯酶（BChE）的研究已进行了几十年,它可以抵制有机磷（OP）神经毒剂,例如甲硫膦酸丙胺乙酯（VX）、甲氟膦酸异丙酯（沙林）和甲氟磷酸异已酯（索曼）等。虽然人们广泛认为BchE对OP暴露具有很大的潜在保护作用,不论是对军人还是对普通平民;但血浆中的BchE浓度为2mg／L,从而使得大范围的纯化及商业化行不通。     

有机磷水解酶及其在传感器应用中的研究进展

有机磷农药的大规模使用对环境造成了严重污染, 同时由于其残留严重威胁着人类健康。有机磷水解酶是一种广泛存在于生物体内的可以催化各种有机磷化合物水解的酶。利用有机磷水解酶制成的生物传感器能够有效检测有机磷农药的残留。文章分别从有机磷水解酶的结构、重组表达以及在生物传感器应用等方面进行了综述, 旨在为有机磷农药的检测和降解提供参考。     

人脯氨肽酶活性中心结构的计算机模拟

氨酰基脯氨酸二肽酶 (脯氨肽酶 )为广泛分布于生物界的细胞内二肽水解酶 .它特异性地水解以脯氨酸或羟脯氨酸为羧基端的二肽 (X Pro) ,而且只对反式肽键有催化活性 .此酶与脯氨酸代谢、胶原蛋白合成及细胞生长有密切关系 .文献报道 ,从Alteromonas细菌中提取的脯氨肽酶有水解梭曼的活性 ,其有机磷酸酐水解酶也有脯氨肽酶活性 .用重组基因表达的人肝脯氨肽酶也同时具有脯氨肽酶活性和水解梭曼的活性 .研究脯氨肽酶活性中心的结构具有重要理论意义和潜在实用价值 .但目前尚无人脯氨肽酶晶体结构的报道 .本文采用蛋白质结构模式识别 (threading)方法对脯氨肽酶的高级结构进行模拟 ,以大肠杆菌甲硫氨酸氨肽酶 (1MAT)为模板 ,模建了人脯氨肽酶C端结构域的空间结构 .通过对模建结构的 3D评估及电荷分布分析 ,对人脯氨肽酶活力中心结构进行了预测 .模建的人脯氨肽酶活性中心位于C端结构域 ,为 6条β折叠围成的一个疏水性口袋 ,外面被 5条α螺旋及一些loop包围 ,活力中心位于疏水结构中央 ,其中有 5个保守氨基酸 ,形成 1个较强的负电荷区 ,周围有 3个较弱的正电荷区域 .实验还发现 ,虽然Mn2 + 或Co2 + 对酶的活性极其重要 ,但对酶蛋白结构的贡献很小 .提示它们可能是在催化反应的电荷转移过程中发挥着重要作用     

库蚊羧酸酯酶研究进展

在昆虫对有机磷杀虫剂抗性的研究中 ,羧酸酯酶 (ｃａｒｂｏｘｙｌｅｓｔｅｒａｓｅｓ)的过量产生是库蚊对有机磷杀虫剂 (ＯＰ)产生抗性的主要机制。羧酸酯酶能够与进入昆虫体内的有机磷杀虫剂快速结合 ,将杀虫剂在到达靶标作用位点前阻隔或降解 ,使其无法发挥原有的杀伤效用。1 .羧酸酯酶的命名库蚊中羧酸酯酶的命名一般根据其水解α 和 β 乙酸萘酯的先后顺序和电泳迁移率不同而定 ;在淀粉电泳中 ,当等量α 乙酸萘酯(α ＮＡ)和 β 乙酸萘酯 (β ＮＡ)同时存在时 ,优先水解α ＮＡ呈蓝色的为酯酶Ａ ,优先水解 β ＮＡ呈红色的为酯酶Ｂ[1] …     

落叶松人工林土壤酸度与有机磷形态的相关性

通过东北东部山地落叶松人工林不同发育阶段土壤酸度变化规律以及与有机磷形态相关关系的研究发现,不同发育阶段落叶松根际土壤水解性总酸度都高于非根际土壤,而交换性酸与总酸度的比值则相反;落叶松由幼龄林到成熟林随林龄的增大根际土壤活性酸(pH值)呈降低趋势,而非根际土壤活性酸(pH值)(除成熟林外)、根际与非根际土壤的交换性酸、土壤交换性铝、土壤水解性总酸度和交换性酸/总酸度的比值随林龄的增大呈显著增大趋势。但由于暗棕壤具有较强的缓冲性能,并且土壤活性酸(pH值)由幼龄林到成熟林随林龄的变化范围为5.27±0.25~5.93±0.12,因此,在落叶松树种适生的范围之内,不需施用石灰调节土壤的酸度。各年龄阶段森林根际土壤酸度与根际土壤有机磷形态相关密切。随着土壤潜性酸度的增加,各年龄阶段森林土壤有机磷总量、中稳性有机磷和高稳性有机磷含量降低。活性有机磷分别与活性酸、水解性总酸度呈显著正相关,其与水解性总酸度相关性随林龄的增大而降低。     

纤维素生物质新型水解技术的研究进展

纤维素生物质水解技术是生物质资源转化的关键技术之一,在传统的酸水解和酶水解技术基础上,近年来出现了一些新型的水解技术,它们一般都具有绿色高效、对环境友好等特点;回顾并综述了纤维素生物质水解技术的最新进展,并对纤维素生物质水解技术的发展研究方向提出了设想.     

人羧酯酶的研究进展

羧酯酶是一类可与有机磷化合物结合且活性受抑制的B－酯酶,分布很广,能水解许多羧酯类、酰胺类、硫酯类物质,其天然底物尚未清楚,故其生理功能仍在研究中,可能与脂质代谢,药物或毒物的生物转化有关．对羧酯酶的一级结构及基因序列的研究表明,羧酯酶是由许多生化特性不同的同工酶组成．     

米曲霉LY-128 广谱有机磷农药水解酶的纯化和鉴定

米曲霉LY-128的培养物经硫酸铵分级沉淀,Sephadex G-100凝胶过滤,DEAE-Sephrose CL-6B和Sephadex G-100层析手段,获得了电泳纯的广谱有机磷农药水解酶.通过SDS-PAGE和IEF电泳测得其分子量为62 kDa,等电点为pH 5.2.该酶的最适反应温度为45℃,最适pH 6.8,在50℃以下及pH6.0～9.5范围内活性稳定.Hg2+、Fe3+、对氯高汞苯甲酸、碘乙酸和N-乙基马来酰亚胺对该酶有强烈的抑制作用,而Cu2+、巯基乙醇、二硫苏糖醇、二硫赤藓糖醇、谷光甘肽和去污剂对酶有不同程度的激活作用.底物的专一性实验表明,该酶不仅可以作用于含P-O键的有机磷农药;而且也能水解含P-S键的有机磷农药.以甲基对硫磷和内吸磷为底物的Km值分别为52μmol、236μmol;Vmax分别为317μmol min-1 mg-1、179 μmol min-1 mg-1;Kcat分别为1152 s-1、650 s-1.     

血液灌流抢救140例急性有机磷农药中毒的护理体会

急性有机磷农药中毒后,有机磷与胆碱酯酶结合形成磷酰化胆碱酯酶,丧失水解乙酰胆碱的能力,导致乙酰胆碱在胆碱能神经突触间隙中蓄积,产生有机磷中毒症状。常用的解毒药物如抗胆碱药物阿托品可阻断节后胆碱能神经支配的效应器上的毒蕈碱受体,对抗乙酰胆碱毒蕈样症状,是治疗有机磷中毒的关键药物,但若中毒量过大,超过了阿托品对抗的极限,则难以挽救病人生命。     

Human butyrylcholinesterase produced in insect cells: huprine-based affinity purification and crystal structure

Butyrylcholinesterase (BChE) is a serine hydrolase that is present in all mammalian tissues. It can accommodate larger substrates or inhibitors than acetylcholinesterase (AChE), the enzyme responsible for hydrolysis of the neurotransmitter acetylcholine in the central nervous system and neuromuscular junctions. AChE is the specific target of organophosphorous pesticides and warfare nerve agents, and BChE is a stoichiometric bioscavenger. Conversion of BChE into a catalytic bioscavenger by rational design or designing reactivators specific to BChE required structural data obtained using a recombinant low-glycosylated human BChE expressed in Chinese hamster ovary cells. This expression system yields ≈ 1 mg of pure enzyme per litre of cell culture. Here, we report an improved expression system using insect cells with a fourfold higher yield for truncated human BChE with all glycosylation sites present. We developed a fast purification protocol for the recombinant protein using huprine-based affinity chromatography, which is superior to the classical procainamide-based affinity. The purified BChE crystallized under different conditions and space group than the recombinant low-glycosylated protein produced in Chinese hamster ovary cells. The crystals diffracted to 2.5 ?. The overall monomer structure is similar to the low-glycosylated structure except for the presence of the additional glycans. Remarkably, the carboxylic acid molecule systematically bound to the catalytic serine in the low-glycosylated structure is also present in this new structure, despite the different expression system, purification protocol and crystallization conditions.     

Comparative Study on the Inhibitory Effect of Caffeic and Chlorogenic Acids on Key Enzymes Linked to Alzheimer’s Disease and Some Pro-oxidant Induced Oxidative Stress in Rats’ Brain-In Vitro

This study sought to investigate and compare the interaction of caffeic acid and chlorogenic acid on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and some pro-oxidants (FeSO₄, sodium nitroprusside and quinolinic acid) induced oxidative stress in rat brain in vitro. The result revealed that caffeic acid and chlorogenic acid inhibited AChE and BChE activities in dose-dependent manner; however, caffeic acid had a higher inhibitory effect on AChE and BChE activities than chlorogenic acid. Combination of the phenolic acids inhibited AChE and BChE activities antagonistically. Furthermore, pro-oxidants such as, FeSO₄, sodium nitroprusside and quinolinic acid caused increase in the malondialdehyde (MDA) contents of the brain which was significantly decreased dose-dependently by the phenolic acids. Inhibition of AChE and BChE activities slows down acetylcholine and butyrylcholine breakdown in the brain. Therefore, one possible mechanism through which the phenolic acids exert their neuroprotective properties is by inhibiting AChE and BChE activities as well as preventing oxidative stress-induced neurodegeneration. However, esterification of caffeic acid with quinic acid producing chlorogenic acid affects these neuroprotective properties.     

Characterization of 12 silent alleles of the human butyrylcholinesterase (BCHE) gene.

The silent phenotype of human butyrylcholinesterase (BChE), present in most human populations in frequencies of approximately 1/100,000, is characterized by the complete absence of BChE activity or by activity <10% of the average levels of the usual phenotype. Heterogeneity in this phenotype has been well established at the phenotypic level, but only a few silent BCHE alleles have been characterized at the DNA level. Twelve silent alleles of the human butyrylcholinesterase gene (BCHE) have been identified in 17 apparently unrelated patients who were selected by their increased sensitivity to the muscle relaxant succinylcholine. All of these alleles are characterized by single nucleotide substitutions or deletions leading to distinct changes in the structure of the BChE enzyme molecule. Nine of the nucleotide substitutions result in the replacement of single amino acid residues. Three of these variants, BCHE*33C, BCHE*198G, and BCHE*201T, produce normal amounts of immunoreactive but enzymatically inactive BChE protein in the plasma. The other six amino acid substitutions, encoded by BCHE*37S, BCHE*125F, BCHE*170E, BCHE*471R, and BCHE*518L, seem to cause reduced expression of BChE protein, and their role in determining the silent phenotype was confirmed by expression in cell culture. The other four silent alleles, BCHE*271STOP, BCHE*500STOP, BCHE*FS6, and BCHE*I2E3-8G, encode BChES truncated at their C-terminus because of premature stop codons caused by nucleotide substitutions, a frame shift, or altered splicing. The large number of different silent BCHE alleles found within a relatively small number of patients shows that the heterogeneity of the silent BChE phenotype is high. The characterization of silent BChE variants will be useful in the study of the structure/function relationship for this and other closely related enzymes.     

Effect of polyethylene glycol modification on the circulatory stability and immunogenicity of recombinant human butyrylcholinesterase

The therapeutic value of human serum butyrylcholinesterase (Hu BChE) as a bioscavenger of chemical warfare agents is due to its high reactivity with organophosphorus compounds and prolonged circulatory stability. Native Hu BChE is mostly tetrameric in form while the enzyme produced using molecular cloning technology is a mixture of tetramers, dimers, and monomers. Previous studies revealed that monomers and dimers of recombinant human (rHu) BChE cleared rapidly from the circulation of mice compared to tetrameric rHu BChE and native Hu BChE, which have mean residence times (MRTs) of 18h and 45h, respectively. It was also shown that polyethylene glycol-20K (PEG) modification of tetrameric rHu BChE prolonged its circulatory stability and bioavailability in vivo. The goal of this study was to determine if modification with PEG could prolong the circulatory stability and eliminate the immunogenicity of monomeric rHu BChE. Monomeric rHu BChE was expressed in human 293A cells using a cDNA lacking the 45 amino acid tetramerization domain from the carboxyl terminus and the adenovirus expression system. The catalytic and inhibitory properties of purified monomeric rHu BChE were similar to those for native Hu BChE and were not affected by PEG modification. As expected, monomeric rHu BChE rapidly cleared from the circulation of mice (MRT=3.2+/-0.3h) while monomeric PEG-rHu BChE demonstrated significant improvement in its bioavailability and circulatory stability in blood (MRT=31.4+/-5.4h). However, a second injection of monomeric PEG-rHu BChE, 28 days after the first, displayed a much shorter MRT=11.6+/-0.4h, and circulating anti-monomeric PEG-rHu BChE antibodies were detected in the blood of mice. These results suggest that PEG modification increased the circulatory stability of monomeric rHu BChE but failed to reduce or eliminate its immunogenicity.     

Development of potent reversible selective inhibitors of butyrylcholinesterase as fluorescent probes

Abstract

Brain butyrylcholinesterase (BChE) is an attractive target for drugs designed for the treatment of Alzheimer’s disease (AD) in its advanced stages. It also potentially represents a biomarker for progression of this disease. Based on the crystal structure of previously described highly potent, reversible, and selective BChE inhibitors, we have developed the fluorescent probes that are selective towards human BChE. The most promising probes also maintain their inhibition of BChE in the low nanomolar range with high selectivity over acetylcholinesterase. Kinetic studies of probes reveal a reversible mixed inhibition mechanism, with binding of these fluorescent probes to both the free and acylated enzyme. Probes show environment-sensitive emission, and additionally, one of them also shows significant enhancement of fluorescence intensity upon binding to the active site of BChE. Finally, the crystal structures of probes in complex with human BChE are reported, which offer an excellent base for further development of this library of compounds.     

Stereoselective inhibition of human, mouse, and horse cholinesterases by bambuterol enantiomers

Bambuterol is a chiral carbamate and a selective inhibitor of butyrylcholinesterase (BChE, EC 3.1.1.8). In order to relate bambuterol selectivity and stereoselectivity of BChE and acetylcholinesterase (AChE, EC 3.1.1.7) of different species, we studied the inhibition of human, mouse, and horse BChE, as well as AChE of human and mouse by (R)- and (S)-bambuterol. AChE and BChE of all studied species were progressively inhibited by both bambuterol enantiomers, with a preference for the (R)-bambuterol whose inhibition rate constants were about five times higher than that of (S)-bambuterol. We observed no significant difference between human and mouse in bambuterol enantiomer BChE inhibition. However, (R)-bambuterol inhibited horse BChE about 14 times slower than human and mouse BChE, and the inhibition rate for (S)-bambuterol was about 18 times slower. Although the primary structure of horse BChE differs from the other two species in 15 amino acids, we presumed that differences in inhibition rates could be attributed to threonine at position 69 located close to the peripheral site of BChE. Since BChE inhibition by bambuterol enantiomers was at least 8000 times faster than that of AChE, both bambuterol enantiomers proved to be selective BChE inhibitors, as was previously shown for racemate.     

Butyrylcholinesterase in lipid metabolism: A new outlook

Cholinesterase enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are traditionally associated with the termination of acetylcholine mediated neural signaling. The fact that these ubiquitous enzymes are also found in tissues not involved in neurotransmission has led to search for alternative functions for these enzymes. Cholinesterases are reported to be involved in many lipid related disease states. Taking into view that lipases and cholinesterases belong to the same enzyme class and by comparing the catalytic sites, we propose a new outlook on the link between BChE and lipid metabolism. The lipogenic substrates of BChE that have recently emerged in contrast to traditional cholinesterase substrates are explained through the hydrolytic capacity of BChE for ghrelin, 4-methyumbelliferyl (4-mu) palmitate, and arachidonoylcholine and through endogenous lipid mediators such as cannabinoids like anandamide and essential fatty acids. The abundance of BChE in brain, intestine, liver, and plasma, tissues with active lipid metabolism, supports the idea that BChE may be involved in lipid hydrolysis. BChE is also regulated by various lipids such as linoleic acid, alpha-linolenic acid or dioctanoylglycerol, whereas AChE is inhibited. The finding that BChE is able to hydrolyze 4-mu palmitate at a pH where lipases are less efficient points to its role as a backup in lipolysis. In diseases such as Alzheimer, in which elevated BChE and impaired lipid levels are observed, the lipolytic activity of BChE might be involved. It is possible to suggest that fatty acids such as 4-mu palmitate, ghrelin, arachidonoylcholine, essential fatty acids, and other related lipid mediators regulate cholinesterases, which could lead to some sort of compensatory mechanism at high lipid concentrations.

     

Polyproline tetramer organizing peptides in fetal bovine serum acetylcholinesterase

Acetylcholinesterase (AChE) in the serum of fetal cow is a tetramer. The related enzyme, butyrylcholinesterase (BChE), in the sera of humans and horse requires polyproline peptides for assembly into tetramers. Our goal was to determine whether soluble tetrameric AChE includes tetramer organizing peptides in its structure. Fetal bovine serum AChE was denatured by boiling to release non-covalently bound peptides. Bulk protein was separated from peptides by filtration and by high performance liquid chromatography. Peptide mass and amino acid sequence of the released peptides were determined by MALDI–TOF–TOF and LTQ-Orbitrap mass spectrometry. Twenty polyproline peptides, divided into 5 families, were identified. The longest peptide contained 25 consecutive prolines and no other amino acid. Other polyproline peptides included one non-proline amino acid, for example serine at the C-terminus of 20 prolines. A search of the mammalian proteome database suggested that this assortment of polyproline peptides originated from at least 5 different precursor proteins, none of which were the ColQ or PRiMA of membrane-anchored AChE. To date, AChE and BChE are the only proteins known that include polyproline tetramer organizing peptides in their tetrameric structure.     

Evolution of acetylcholinesterase and butyrylcholinesterase in the vertebrates: an atypical butyrylcholinesterase from the Medaka Oryzias latipes

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are thought to be the result of a gene duplication event early in vertebrate evolution. To learn more about the evolution of these enzymes, we expressed in vitro, characterized, and modeled a recombinant cholinesterase (ChE) from a teleost, the medaka Oryzias latipes. In addition to AChE, O. latipes has a ChE that is different from either vertebrate AChE or BChE, which we are classifying as an atypical BChE, and which may resemble a transitional form between the two. Of the fourteen aromatic amino acids in the catalytic gorge of vertebrate AChE, ten are conserved in the atypical BChE of O. latipes; by contrast, only eight are conserved in vertebrate BChE. Notably, the atypical BChE has one phenylalanine in its acyl pocket, while AChE has two and BChE none. These substitutions could account for the intermediate nature of this atypical BChE. Molecular modeling supports this proposal. The atypical BChE hydrolyzes acetylthiocholine (ATCh) and propionylthiocholine (PTCh) preferentially but butyrylthiocholine (BTCh) to a considerable extent, which is different from the substrate specificity of AChE or BChE. The enzyme shows substrate inhibition with the two smaller substrates but not with the larger substrate BTCh. In comparison, AChE exhibits substrate inhibition, while BChE does not, but may instead show substrate activation. The atypical BChE from O. latipes also shows a mixed pattern of inhibition. It is effectively inhibited by physostigmine, typical of all ChEs. However, although the atypical BChE is efficiently inhibited by the BChE-specific inhibitor ethopropazine, it is not by another BChE inhibitor, iso-OMPA, nor by the AChE-specific inhibitor BW284c51. The atypical BChE is found as a glycophosphatidylinositol-anchored (GPI-anchored) amphiphilic dimer (G(2) (a)), which is unusual for any BChE. We classify the enzyme as an atypical BChE and discuss its implications for the evolution of AChE and BChE and for ecotoxicology.