The protein acetylcholinesterase (AChE) has been isolated from the electroplax tissue of the electric eel (Electrophorus electricus) and purified by affinity chromatography. Working with fresh tissue, the structural stability of this kind of preparation towards proteolysis and/or autolysis has been investigated. Gel electrophoresis of the purified enzyme, in the presence of sodium dodecylsulphate and dithiothreitol, shows predominantly one component at 80,000 molecular weight. However, gels run at various times after purification demonstrate that the 80,000 polypeptide is susceptible to cleavage generating peptides of 55,000, 28,000 and 25,000 molecular weight. Evidence is presented to show that AChE is composed of four identical subunits arranged as a dimer of dimers (α₂)₂. Incubation of the freshly affinity purified AChE with trypsinis shown to mimic the cleavage of the 80,000 subunit by endogeneous protease. Sucrose gradient centrifugation of purified AChE shows it to be composed of two forms characterised by their sedimentation coefficients of 18S and 14S which upon proteolysis convert to a globular 11S form. Furthermore conversion of the 'native' molecular forms to the globular form occurs faster than proteolytic cleavage of the catalytic subunit. Some chemical modification of the protein is described in the last section of the thesis. The enzyme has been labelled, by two different and complementary methods, so as to incorporate radioactive iodide¹²⁵, I. Of particular interest is the result observed with an enzymatic, lacto-peroxidase iodination of the 11S form of the enzyme which shows that greater than 90% of the label is incorporated into the low molecular weight components of the subunit. The other results of the two iodination methods are described and discussed. Finally, an appendix describing the characterisation of AChE via isokinetic sucrose gradients in included. / Science, Faculty of / Chemistry, Department of / Graduate

Involvement of lipid in the activity of mammalian erythrocyte acetylcholinesterase (AChE) has been proposed by various workers. In bovine erythrocyte AChE a tightly bound fraction of cardiolipin (CL) was proposed to be involved in the modulation of AChE catalytic activity. Methods previously used for the isolation of CL resulted in enzyme denaturation. In the present study various methods for the separation of CL under non-denaturing conditions have been investigated. It was reported earlier that all the lipoprotein forms of the enzyme containing CL showed a biphasic Arrhenius plot with a break around 20°C. It was suggested that treatment of the enzyme with 1.8M sodium chloride, 2mM sodium phosphate, pH 7.4 or IM sodium bicarbonate pH 8-10, caused dissociation of cardiolipin and it was accompanied by abolition of the Arrhenius plot break. Methods used for the separation of CL from AChE were based on the difference in size and density between the two components. Enzyme was treated with "high salt" conditions which were postulated to cause dissociation of CL. The resulting mixture was passed through a Sephadex gel column so that CL can be separated from the enzyme because of its size difference. The enzyme obtained from the Sephadex gel column gave a partial specific volume of 0.81 ml/g, which is higher than that expected from the amino acid composition of the protein, indicating that CL is still bound to the enzyme. In another experiment ("flotation experiment") an attempt was made to separate the dissociated CL from the enzyme on a sucrose gradient, based on the density difference between the phopholipid and protein. Arrhenius plots were obtained at different time intervals on the enzyme recovered from the sucrose gradient. A linear Arrhenius plot was observed after 24 h. Storage of the enzyme for 5 to 8 days gave rise to a distinct break in the Arrhenius plot. The reapperarance of the break was observed even when the centrifugation was done in the presence of 0.09% Triton X-100. This was interpreted to indicate that endogeneous CL was bound to the enzyme through ionic and hydrophobic interaction. "High salt" treatment may abolish the ionic interaction, causing "functional dissociation" of CL (as shown by disappearance of the Arrhenius plot break) but simultaneous strengthening of the hydrophobic interactions may account for the reappearance of the break. The next method attempted for the separation of CL was based on the principle that, if enzyme could be bound to a solid support, then washing of the enzyme with chaotropic agents, detergents and "high salt" may result in the release of CL. The suitability of the N-methylacridinium (MAC) affinity column as a solid support for this enzyme was investigated. The choice of MAC as an affinity ligand was based on the recent reports regarding its suitability as an affinity ligand for purification of eel and pig brain AChE. The elution profile of the enzyme in 0.IM NaCl, 20mM sodium phosphate, pH 7.4 at different ligand (MAC) concentrations indicated that a minimum of 2.8 umole/ml gel required for sufficient retention of the enzyme. As the affinity of the ligand for the enzyme will further decrease with increasing ionic strength, the MAC affinity column, is unsuitable as a solid support for bovine erythrocyte AChE. It was found that the lower retention of the bovine erythrocyte AChE compared to the eel enzyme on the MAC affinity columns was due to the lower affinity of the former for the ligand, rather than to any structural difference or a different mode of binding. Preliminary findings suggest that CL dissociation did not alter the affinity of the enzyme for the ligand. Finally, as a primary requirement for the preparation of large quantities', of pure AChE, so that CL can be exchanged by a detergent exchange method, various steps for the purification of the native forms of AChE by a detergent free method have been characterized. The following findings were made. Butanol treatment enhanced the enzyme release .from the membrane, from 40 to 80 percent, by extracting the "mobile" phospholipids. The extraction of AChE can be increased by increasing the ionic strength of the medium and by calcium chelation. Purification of the above enzyme can be acheived by affinity purification but optimum conditions required for the above purification are still under investigation. Characterization of the molecular forms of the enzyme on sucrose density gradient indicates extensive aggregation at low ionic strength, while a lower degree of aggregation with a prominant 11S peak was observed in the presence of 0.IM sodium chloride, 20mM sodium phosphate. / Pharmaceutical Sciences, Faculty of / Graduate

Acetylcholinesterase (AChE, E.C. 3.1.1.7) from 1-day chick brain was enriched over 2,000-fold by N-methylacridinium affinity column. Using this preparation as immunogen, two monoclonal antibodies (mAbs), 1E2 and 3D10, were isolated. Both mAbs react with all molecular forms of AChE from chick but not with butyrylcholinesterases (BuChE, E.C. 3.1.1.8). The mAb, 1E2, was used to immunopurify chick brain globular and muscle asymmetric AChE to homogeneity. The purified brain AChE showed a specific activity of 2,200 U/mg of protein, and it appeared to be a hydrophobic tetramer with a subunit mass of 105 kDa. The purified muscle asymmetric AChE has a specific activity of 1,100 U/mg of protein, it exhibits catalytic and inhibition properties characteristic of both AChE and BuChE and contains three distinct subunits with an apparent size of 110 kDa, 72 kDa and 58 kDa in the ratio 2:2:1. The discovery of an AChE/BuChE hybrid asymmetric form has been further supported by: (1) the identification of active site properties of AChE in the 110kDa subunit and of BuChE in the 72-kDa subunit, (2) the purification and precipitation of both activities by a BuChE-specific mAb (7D11), and (3) the evidence that all subunits are bound in the asymmetric form by disulphide bonds. The 58-kDa subunit is the only one that is sensitive to digestion with purified collagenase; it carries the collagenous 'tail'.

This thesis describes the isolation and characterization of four steroidal alkaloids from Buxus macowanii. Phytochemical investigation of Buxus macowanii resulted in the isolation of two novel steroidal alkaloids, Nb-demethyl-6-deoxy-16-acetoxy O2-natafuranamin (112), and 6-deoxy-16-acetoxy O10-natafuranamin (113) alongside two known steroidal alkaloids, cycloprotobuxine-D (114), and cycloprotobuxine-F (115). Compounds 114 and 115 have been isolated for the first time from B. macowanii. Structure of compounds 112-115 was elucidated with aid of UV, IR, mass, and 1D and 2D NMR spectroscopy. These compounds showed different level of anti-AChE activity. Among all the isolates, compound 112 was found to be significantly active against AChE with an IC50 value of 4.7 µM. The bioactivity of this new compound nearly comparable to those of huperzine (IC50 = 1.7 µM) and O2-natafuranamine (IC50 = 3.0 µM).

乙酰膽鹼酯酶主要存在於神經肌肉接頭處和中樞神經系統的膽鹼能突觸處，是神經遞質傳遞過程中極其重要的膽鹼水解酶。研究表明，阿茲海默病人的大腦通常呈現出乙酰膽鹼酯酶的異常表達和分佈，並伴隨著β澱粉樣蛋白的沉澱。目前，乙酰膽鹼酯酶抑製劑是治療阿茲海默症的主要臨床藥物。 / 在本研究中，我們利用Bac-to-Bac 桿狀病毒表達系統分別使人類和雞泡魚的重組乙酰膽鹼酯酶基因在家蠶幼蟲裡得到了高效的表達。我們將乙酰膽鹼酯酶的cDNA序列克隆到pFastBac{U+2122} Dual質粒的多角體蛋白啟動子下游。為了易於監控蛋白表達水平，橙色熒光蛋白的cDNA序列也被克隆到同一個質粒的p10啟動子下游。此外，我們將多聚組氨酸標籤加在了乙酰膽鹼酯酶基因的碳端，從而使蛋白的純化效率得到了顯著提高。我們通過皮下注射含有乙酰膽鹼酯酶的重組bacmid對五齡期的家蠶幼蟲進行了病毒轉染。感染後約4-7天，重組乙酰膽鹼酯酶在蠶蟲內成功得到了表達。酶促反應動力學研究表明，重組乙酰膽鹼酯酶的活性與來自相同物種的天然乙酰膽鹼酯酶基本相似。這種高效率、低成本、高產量的蛋白表達方法可以為我們提供大量的重組乙酰膽鹼酯酶，用於體外篩選治療阿茲海默症的乙酰膽鹼酯酶抑製劑。 / 隨著對阿茲海默症分子學水平上的進一步了解，研究提出乙酰膽鹼酯酶可能通過外周陰離子位點誘導β澱粉樣多肽聚集, 從而形成澱粉樣纖維。因此，理想的乙酰膽鹼酯酶抑製劑應該既有抑制乙酰膽鹼酯酶的活性，又可以對抗β澱粉樣蛋白沉澱的毒性, 從而達到神經保護的作用。因此，我們採用AutoDock Vina軟件對ZINC數據庫內的天然化合物進行了兩輪虛擬篩選，篩選出的化合物理論上是可以同時作用於催化位點和外周陰離子位點。接下來，我們將對候選化合物進行體外驗證。 / Acetylcholinesterase (AChE: EC 3.1.1.7) is the acetylcholine-hydrolyzing enzyme that plays an essential role on cholinergic neurotransmission at the synapses of the brain and at the neuromuscular junctions. Abnormal expression and localization of AChE have been observed together with Aβ deposits in the brain of Alzheimer’s disease (AD) patient. Currently, AChE inhibitors are clinically used as drugs for AD treatment. / In this study, we demonstrated high-level expressions of functional recombinant human AChE and Tetraodon nigroviridis AChE using Bac-to-Bac baculovirus expression system in silkworm Bombyx mori larvae. The cDNA of AChE was cloned into the polyhedrin (PH) promoter of the plasmid pFastBac{U+2122} Dual. To monitor the level of expression, the cDNA coding for an orange fluorescent protein (OFP2) was cloned downstream to the p10 promoter of the same vector. We engineered a polyhistidine-tag (His-tag) tail to the C-terminal of each AChE gene to facilitate the purification. Transfection was carried out by subcutaneous injection of the recombinant bacmid DNA containing the AChE gene into the silkworm larvae of 5th instar. Approximately 4-7 days of post-infection, the recombinant AChE was expressed in the hemolymph of infected larvae. The kinetic studies showed that the biological activities of the recombinant AChEs were comparable to that of natural ones from other sources. This rapid, low-cost, and high yield production method could provide us sufficient amount of recombinant AChE for in vitro screening of AChE inhibitors for AD treatment. / Further advances in understanding the molecular basis of AD have proposed that AChE promote the assembly of Aβ peptide into amyloid fibrils through interaction at the peripheral anionic site of AChE. Consequently, new classes of AChE inhibitors are expected to be able to inhibit the active site of AChE and, at the same time, to protect neurons from Aβ toxicity. Therefore, we applied two rounds of virtual screening of ZINC database using AutoDock Vina to obtain new potential inhibitors which might be able to targeting both of the active and peripheral sites of AChE. The compounds with good performances in both of the two rounds of screening would be validated by the sequential in vitro tests. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Shuo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 105-124). / Abstracts also in Chinese. / Acknowledgements --- p.I / Abstracts (English) --- p.II / Abstracts (Chinese) --- p.IV / Table of Contents --- p.VI / List of Abbreviations --- p.IX / List of Figures --- p.XII / List of Tables --- p.XIV / Chapter Chapter 1 Introduction --- p.1 / Chapter 1.1 --- Acetylcholine mediated neutotransmission in nervous system --- p.1 / Chapter 1.2 --- Acetylcholineterase --- p.2 / Chapter 1.3 --- Comparison of AChE and BChE --- p.3 / Chapter 1.4 --- Molecular sturcture of AChE --- p.5 / Chapter 1.5 --- Molecular diversity of AChE --- p.7 / Chapter 1.5.1 --- Regulation at transcriptional level --- p.8 / Chapter 1.5.2 --- Regulation at post-transcriptional level --- p.11 / Chapter 1.5.3 --- Regulation at post-translational level --- p.12 / Chapter 1.6 --- Classic functions of AChE --- p.15 / Chapter 1.7 --- Non-classic functions of AChE --- p.18 / Chapter 1.8 --- Diseases associated with AChE --- p.19 / Chapter 1.8.1 --- Myasthenia gravis --- p.19 / Chapter 1.8.2 --- Alzheimer's disease --- p.20 / Chapter 1.8.2.1 --- Pathogenesis of AD --- p.20 / Chapter 1.8.2.2 --- Treatments for AD --- p.22 / Chapter 1.9 --- Silkworm larvae as biofactory for protein expression --- p.24 / Chapter 1.10 --- Traditional baculovirus expression system --- p.26 / Chapter 1.11 --- Bac-to-Bac baculovirus expression system --- p.29 / Chapter 1.12 --- Virtual screening with AutoDock Vina --- p.29 / Chapter 1.13 --- Project overview and the aim of study --- p.31 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.33 / Chapter 2.1.1 --- Chemicals and Reagents --- p.33 / Chapter 2.1.2 --- Primers --- p.35 / Chapter 2.1.3 --- Antibodies --- p.35 / Chapter 2.1.4 --- Silkworms --- p.35 / Chapter 2.2 --- Methods --- p.36 / Chapter 2.2.1 --- Construction of the expression cassette --- p.36 / Chapter 2.2.1.1 --- Preparation of E.coli competent cells --- p.36 / Chapter 2.2.1.2 --- Transformation --- p.36 / Chapter 2.2.1.3 --- Agarose gel electrophoresis --- p.37 / Chapter 2.2.1.4 --- Gene clean --- p.37 / Chapter 2.2.1.5 --- Subcloning of target genes --- p.38 / Chapter 2.2.1.6 --- Plasmid DNA extraction --- p.40 / Chapter 2.2.1.7 --- Quantification of plasmid DNA by spectrophotometer --- p.41 / Chapter 2.2.1.8 --- Plasmid DNA sequencing --- p.41 / Chapter 2.2.2 --- Generation of recombinant bacmid DNA --- p.42 / Chapter 2.2.2.1 --- Transposition --- p.42 / Chapter 2.2.2.2 --- White/Blue screening --- p.42 / Chapter 2.2.2.3 --- Extraction of recombinant bacmid DNA --- p.42 / Chapter 2.2.2.4 --- Analysis of recombinant bacmid DNA by PCR --- p.44 / Chapter 2.2.3 --- Transfection of silkworm larvae --- p.45 / Chapter 2.2.3.1 --- Raising silkworm larvae --- p.45 / Chapter 2.2.3.2 --- Preparation of transfecting solution --- p.45 / Chapter 2.2.3.3 --- Transfection of silkworm larvae --- p.45 / Chapter 2.2.3.4 --- Collection of hemolymph after protein expression --- p.46 / Chapter 2.2.3.5 --- Oral infection of sikworm larvae --- p.46 / Chapter 2.2.4 --- Purification of AChE --- p.47 / Chapter 2.2.4.1 --- Nickel-chelating afinity chromatography --- p. 47 / Chapter 2.2.4.2 --- Determination of protein concenttration by BCA assay --- p.47 / Chapter 2.2.4.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.48 / Chapter 2.2.4.4 --- Western blot --- p.49 / Chapter 2.2.5 --- Kinetic analysis of AChE --- p.50 / Chapter 2.2.5.1 --- Ellman assay --- p.50 / Chapter 2.2.5.2 --- Curve fitting --- p.51 / Chapter 2.2.6 --- Virtual screening --- p.51 / Chapter Chapter 3 --- Expression of recombinant AChEs in silkworm larvae --- p.55 / Chapter 3.1 --- Construction of the expression cassette --- p.55 / Chapter 3.1.1 --- Human AChE and Tetraodon nigroviridis AChE --- p.55 / Chapter 3.1.2 --- Amplification of target genes from the parent vectors --- p.56 / Chapter 3.1.3 --- Insertion of target genes into pFastBac Dual --- p.58 / Chapter 3.2 --- Generation of recombinant bacmid DNA --- p.60 / Chapter 3.2.1 --- Phenotype verification --- p.60 / Chapter 3.2.2 --- PCR analysis of the recoombinant bacmid DNA --- p.64 / Chapter 3.3 --- Expression of AChE in silkworm larvae --- p.66 / Chapter 3.3.1 --- Raising silkworms --- p.66 / Chapter 3.3.2 --- High-level expression of AChE in silkworm larvae --- p.68 / Chapter 3.4 --- Oral infeciton --- p.72 / Chapter Chapter 4 --- Analysis of the recombinant AChEs --- p.73 / Chapter 4.1 --- Purification of recombinant AChEs by nickel-chelating affinity chromatography --- p.73 / Chapter 4.2 --- SDS-PAGE and western blot analysis of the recombinant AChEs --- p.76 / Chapter 4.3 --- Kinetic studies of recombinant AChEs --- p.79 / Chapter 4.4 --- Virtual screening --- p.84 / Chapter Chapter 5 --- Discussion and conclusion --- p.95 / Chapter 5.1 --- Demonstration of high-level expression of recombinant AChEs by Bac-to-Bac baculovirus expression system --- p.95 / Chapter 5.2 --- Oral infection of silkworm larvae --- p.98 / Chapter 5.3 --- Characterization of recombinant AChEs --- p.98 / Chapter 5.4 --- Discovery of new AChE inhibitors by virtual screening --- p.100 / Chapter 5.5 --- Future works --- p.101 / Chapter 5.6 --- Other applications --- p.102 / Chapter 5.7 --- Conclusion --- p.102 / References --- p.104 / Appendix I --- p.125 / Appendix II --- p.127 / Appendix III --- p.129

published_or_final_version / Biochemistry / Master / Master of Philosophy

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Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Student: Jakub Jeřábek Supervisors: Prof. PharmDr. Martin Doležal, Ph.D. Prof. Maria Laura Bolognesi Title of Thesis: Design and Synthesis of Hybrid Compounds Based on Tacrine/Resveratrol Derivatives Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder, in which a progressive dementia appears. The cause of AD is currently unknown, however, scientific research has revealed several pathological hallmarks - β-amyloid plaques and neurofibrillary tangles. These changes cause gradual disintegration of nerve cells and they change the metabolism in the brain. The current drugs are not able to treat the cause of the disease, being able only to delay the onset of severe symptoms. The basic drugs for AD treatment are acetylcholinesterase (AChE, E.C. 3.1.1.7) inhibitors and, more recently approved, N-methyl- D-aspartate (NMDA) receptor antagonist memantine. These drugs are able to increase cholinergic activity or preventing glutamate excitotoxicity in the patient's brain, thus improving cognitive functions and delaying severe stages of the disease. One of the emerging approaches in drug synthesis represents multi-target-directed ligands (MTDLs). Apart from the ability...

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 195-210). Also available in electronic version.

This document describes research conducted during 2009-2010 in the Kerman Group laboratory at the University of Toronto Scarborough to investigate the application of electrochemical techniques for the detection of acetylcholinesterase inhibitors in aqueous samples. Two main projects were completed and are discussed herein. The first project demonstrated that the new unmodified, nanostructured gold disposable electrochemical printed (DEP) chips produced by BioDevice Technology can compete with surface-modified electrode configurations to detect trace concentrations of insecticides. This was achieved through the measurement of acetylcholinesterase-catalyzed production of thiocholine after incubation of the enzyme with low concentrations of paraoxon (10 ppb) and carbofuran (8 ppb). The second project featured the novel application of a glassy carbon (GC) electrode to monitor the changes in availability of Thioflavin T (ThT) for oxidation at the electrode surface, which is non-linearly modulated by the presence of acetylcholinesterase and the enzyme’s pre-treatment with trace concentrations of paraoxon and carbachol.