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%。     

Purification and characterization of acetylcholinesterase from cotton aphid (Aphis gossypii Glover)

A simple and effective method was set up to purify acetylcholinesterase (AChE, EC3.1.1.7) from the cotton aphid, Aphis gossypii Glover. The procedure involved filtration on a sephadex G-25 column, separation with sephadex G-200 and procainamide affinity column. AChE from both susceptible and resistant strains were purified to a single band as resolved on denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity increased by 35,100- and 33,680-fold with a yield of 30.3 and 29.8%, respectively. The molecular mass of the purified AChE was about 63,500 Dalton as determined by SDS-PAGE. However, three bands resolved on PAGE gel electrophoresis, leading to the inference that native AChE exists in three forms. The optimum conditions for measuring the activity of purified AChE with kinetic method were 0.02M phosphate buffer, pH7.2, 0.02 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and 25 degrees C. Investigation also revealed that crude extract and purified AChE had different kinetic characteristics and inhibitory properties. They responded differently to varied DTNB, ATChI, and phosphate buffer ion concentrations, as well as pH, temperature, and inhibitors. The purified AChE was more sensitive to eserine, methamidophos, and pirimicarb. Especially for resistant aphids, the sensitivity of purified AChE to methamidophos and pirimicarb was enhanced 6.43 and 11.73 times, respectively. We infer that one or more factors in the crude extract from the resistance strain have more influence on AChE sensitivity. Further study is needed to investigate the basis of these observations.     

Expression of the housefly acetylcholinesterase in a bioreactor and its potential application in the detection of pesticide residues

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.     

Monoclonal Antibodies to Rabbit Brain Acetylcholinesterase: Selective Enzyme Inhibition, Differential Affinity for Enzyme Forms, and Cross-Reactivity with Other Mammalian Cholinesterases

Eleven unique monoclonal IgG antibodies were raised against rabbit brain acetylcholinesterase (AChE, EC 3.1.1.7), purified to electrophoretic homogeneity by a two-step procedure involving immunoaffinity chromatography. The apparent dissociation constants of these antibodies for rabbit AChE ranged from about 10 nM to more than 100 nM (assuming one binding site per catalytic subunit). Species cross-reactivity was investigated with crude brain extracts from rabbit, rat, mouse cat, guinea pig, and human. One antibody bound rabbit AChE exclusively; most bound AChE from three or four species; two bound enzyme from all species tested. Identical, moderate affinity for rat and mouse brain AChE was displayed by two antibodies; two others were able to distinguish between these similar antigens. Nine of the antibodies had lowered affinity for AChE in the presence of 1 M NaCl, but two were salt resistant. Analysis of mutual interferences in AChE binding suggested that certain of the antibodies were competing for nearby epitopes on the AChE surface. One antibody was a potent AChE inhibitor (IC50 = 10(-8) M), blocking up to 90% of the enzyme activity. Most of the antibodies were less able to bind the readily soluble AChE of detergent-free brain extracts than the AChE which required detergent for solubilization. The extreme case, an antibody that was unable to recognize nearly half of the "soluble" AChE, was suspected of lacking affinity for the hydrophilic enzyme form.     

Inhibition of two different cholinesterases by tacrine

Kinetic parameters of the effect of tacrine as a cholinesterase inhibitor have been studied in two different sources: snake venom (Bungarus sindanus) acetylcholinesterase (AChE) and human serum butyrylcholinesterase (BChE). Tacrine inhibited both venom acetylcholinesterase (AChE) as well as human serum butyrylcholinesterase (BChE) in a concentration-dependent manner. Kinetic studies indicated that the nature of inhibition was mixed for both enzymes, i.e. Km values increase and Vmax decrease with the increase of the tacrine concentration. The calculated IC50 for snake venom and for human serum were 31 and 25.6 nM, respectively. Ki was observed to be 13 nM for venom acetylcholinesterase (AChE) and 12 nM for serum butyrylcholinesterase (BChE). KI (constant of AChE-ASCh-tacrine complex into AChE-ASCh complex and tacrine) was estimated to be 20 nM for venom and 10 nM for serum butyrylcholinesterase (BChE), while the gammaKm (dissociation constant of AChE-ASCh-tacrine complex into AChE-tacrine complex and ASCh) were 0.086 and 0.147 mM for snake venom AChE and serum BChE, respectively. The present results suggest that this therapeutic agent used for the treatment of Alzheimer's disease can also be considered an inhibitor of snake venom and human serum butyrylcholinesterase. Values of Ki and KI show that tacrine had more affinity with these enzymes as compared with other cholinesterases from the literature.     

Substance P in Human Plasma Is Degraded by Dipeptidyl Peptidase IV, Not by Cholinesterase

Human serum cleaves two dipeptides from the N-terminus of the neurohormone substance P. It has been suggested that this degrading activity is inherent to serum cholinesterase. We oppose this, because it turned out that highly purified serum cholinesterase contains traces of dipeptidyl peptidase IV, an enzyme known to attack the N-terminus of substance P. The peptidase is incompletely separated from cholinesterase during the procainamide-gel affinity chromatography as the last step of the usual purification procedure. Physostigmine completely inhibits the hydrolysis of butyrylthiocholine by such purified cholinesterase preparations, but not their substance P-degrading activity. Vice versa, epsilon-carbobenzoxy-lysylproline, an inhibitor of dipeptidyl peptidase IV, inhibits the peptidase activity of these preparations more than their esterase activity. After rechromatography on procainamide gel the peptidase is completely separated and the remaining cholinesterase has lost its substance P-degrading activity. We conclude that the N-terminal region of substance P is not degraded by cholinesterase but by the contaminating dipeptidyl peptidase IV, a different serine enzyme.     

Verification of therapeutic levels of procainamide and N-acetyl- procainamide in ventricular arrhythmia]

Therapeutical efficacy was clinically evaluated in 21 patients with ventricular cardiac arrhythmias. The drug was given orally with preceded intramuscular dose. Therapeutic effect was verified by the measurements of procainamide and N-acetylprocainamide concentrations in blood serum to determine the minimal effective concentration of the drug required to obtain satisfactory antiarrhythmic effect. Procainamide proved effective in cardiac arrhythmias in 14 patients (66.7%) with statistical significance in the acute myocardial infarctions; blood serum procainamide plus N-acetylprocainamide levels being were below the therapeutical range. The poor correlation of the dose of the drug and respective procainamide, N-acetylprocainamide concentrations in blood was observed. Relationship of the therapeutical effects blood serum level of the drug should be estimated basing of the assays of both procainamide and N-acetylprocainamide .     

Separation in a single step by affinity chromatography of cholinesterases differing in subunit number.

We describe an affinity chromatography method in which dimethylaminoethylbenzoic acid-Sepharose 4B is used, making it possible to separate in one step the molecular forms of globular acetylcholinesterase (AChE, EC 3.1.1.7) or butyrylcholinesterase (ChE, EC 3.1.1.8). A crude extract containing these enzymes was deposited onto the chromatography gel, washed, and eluted by a linear gradient of tetramethylammonium chloride (0-0.3 M). With rat brain AChE, two well-separated peaks were eluted in the presence of 1% Triton X-100; the first peak corresponded to 4 S forms and the second to 11 S forms. This separation was very efficient for salt-soluble activity and less efficient for the detergent-soluble AChE. In this case, the 4 S peak represented only 6.5% of total detergent-soluble activity and was cross-contaminated by the 11 S form. Rat serum ChE was efficiently separated into two peaks of 7 S and 11 S. This method could potentially be adapted to separate other multimeric proteins with varying numbers of affinity sites.     

Purification and properties of hydrophilic dimers of acetylcholinesterase from mouse erythrocytes

Differences in the glycosylation of acetylcholinesterase (AChE) subunits which form the dimers of mouse erythrocyte and a suitable procedure to purify the enzyme by affinity chromatography in edrophonium-Sepharose are described. AChE was extracted ( approximately 80%) from erythrocytes with Triton X-100 and sedimentation analyses showed the existence of amphiphilic AChE dimers in the extract. The AChE dimers were converted into monomers by reducing the disulfide bond which links the enzyme subunits. Lectin interaction studies revealed that most of the dimers were bound by concanavalin A (Con A) (90-95%), Lens culinaris agglutinin (LCA) (90-95%), and wheat germ (Triticum vulgaris) agglutinin (WGA) (70-75%), and a small fraction by Ricinus communis agglutinin (RCA(120)) (25-30%). The lower level of binding of the AChE monomers with WGA (55-60%), and especially with RCA (10-15%), with respect to the dimers, reflected heterogeneity in the sugar composition of the glycans linked to each AChE subunit in dimers. Forty per cent of the amphiphilic AChE dimers lost the glycosylphosphatidylinositol (GPI) and, therefore, were converted into hydrophilic forms, by incubation with phosphatidylinositol-specific phospholipase C (PIPLC), which permitted their separation from the amphiphilic variants in octyl-Sepharose. Only the hydrophilic dimers, either isolated or mixed with the amphiphilic forms, were bound by edrophonium-Sepharose, which allowed their purification (4800-fold) with a specific activity of 7700 U/mg protein. The identification of a single protein band of 66 kDa in gel electrophoresis demonstrates that the procedure can be used for the purification of GPI-anchored AChE, providing that the attached glycolipid domain is susceptible to PIPLC.     

Butyrylcholinesterase Is Complexed with Transferrin in Chicken Serum

The function of the enzyme butyrylcholinesterase (BChE) both in serum and in brain is unclear. In serum, BChE has been found complexed with several biomedically relevant proteins, with which it could function in concert. Here, the existence of a similar complex formed between BChE and sero-transferrin from adult chicken serum was elucidated. In order to identify both proteins unequivocally, we improved methods to highly purify the 81-kDa BChE and the coisolated 75-kDa transferrin, which then allowed us to tryptically digest and sequence the resulting peptides. The sequences as revealed for BChE peptides were highly identical to mammalian BChEs. A tight complex formation between the two proteins could be established (a) since transferrin is coisolated along with BChE over three steps including procainamide affinity chromatography, while transferrin alone is not bound to this affinity column, and (b) since imunoprecipitation experiments of whole serum with a transferrin-specific antiserum allows us to detect BChE in the precipitate with the BChE-specific monoclonal antibody 7D11. The possible biomedical implications of a complex between transferrin and BChE which here has been shown to exist in chicken serum are briefly discussed.     

Two-Step Immunoaffinity Purification of Acetylcholinesterase from Rabbit Brain

Acetylcholinesterase (AChE; EC 3.1.1.7) extracted in 1% Triton X-100 from rabbit brain was purified 2,000-fold by chromatography on agarose conjugated with a monoclonal antibody directed against human red blood cell cholinesterase. After elution from the immunoadsorbent with pH 11 buffer, the preparation was purified further by affinity chromatography on phenyltrimethylammonium-Sepharose 4B with decamethonium elution. Overall yield of purified enzyme was 37% of the AChE originally solubilized, with a specific activity of 2,950 units/mg protein. Electrophoresis under reducing conditions in 7.5% sodium dodecyl sulfate polyacrylamide gels revealed only one silver-staining polypeptide band. A streamlined purification procedure enabled the isolation of electrophoretically homogeneous AChE to be completed in fewer than 7 days, at yields exceeding 50%. Electrophoretic analysis of purified AChE indicated an apparent MW of 71,000 for the monomeric subunit. Gel filtration and sucrose density gradient centrifugation in the presence of Triton X-100 showed little difference between the properties of the native and the purified enzyme. The molecular mass of the main species was estimated from the gel filtration and sedimentation data to be 280,000 daltons. Kinetic parameters of the purified protein (Km = 0.16 +/- 0.01 mM) were close to those of the native enzyme (Km = 0.12 +/- 0.01 mM) when examined with acetylthiocholine iodide as substrate. The two-step immunopurification procedure presented in this communication offers a convenient route to homogeneous neural AChE in quantities useful for detailed biochemical and immunochemical study.     

Drosophila acetylcholinesterase: demonstration of a glycoinositol phospholipid anchor and an endogenous proteolytic cleavage

The presence of a glycoinositol phospholipid anchor in Drosophila acetylcholinesterase (AChE) was shown by several criteria. Chemical analysis of highly purified Drosophila AChE demonstrated approximately one residue of inositol per enzyme subunit. Selective cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) was tested with Drosophila AChE radiolabeled by the photoactivatable affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID), a reagent that specifically labels the lipid moiety of glycoinositol phospholipid-anchored proteins. Digestion with PI-PLC released 75% of this radiolabel from the protein. Gel electrophoresis of Drosophila AChE in sodium dodecyl sulfate indicated prominent 55- and 16-kDa bands and a faint 70-kDa band. The [125I]TID label was localized on the 55-kDa fragment, suggesting that this fragment is the C-terminal portion of the protein. In support of this conclusion, a sensitive microsequencing procedure that involved manual Edman degradation combined with radiomethylation was used to determine residues 2-5 of the 16-kDa fragment. Comparison with the Drosophila AChE cDNA sequence [Hall, L.M.C., & Spierer, P. (1986) EMBO J. 5, 2949-2954] confirmed that the 16-kDa fragment includes the N-terminus of AChE. Furthermore, the position of the N-terminal amino acid of the mature Drosophila AChE is closely homologous to that of Torpedo AChE. The presence of radiomethylatable ethanolamine in both 16- and 55-kDa fragments was also confirmed. Thus, Drosophila AChE may include a second posttranslational modification involving ethanolamine.     

Purifcation and properties of multiple forms of brain acetylcholinesterase (EC 3.1.1.7)

—Approximately 70 per cent of the total AChE of bovine brain tissue was solubilized by repeated homogenization and centrifugation in 0.32 m sucrose containing EDTA. After ammonium sulphate fractionation, application of the enzyme preparation to an agarose affinity gel column effected a 700-fold purification. Subsequent molecular filtration separated three active forms of AChE with molecular weights of 130,000, 270,000 and 390,000 with an average specific activity of 575 mmol of acetylthiocholine hydrolysed/mg of protein/h. The complete procedure represented an approximate 23,000-fold purification of the enzyme from that in the original tissue homogenate. The three forms of AChE exhibited certain differences in properties, including apparent K_m values, pH optima and sensitivity to inhibitory agents. Ancillary studies on less purified enzyme preparations by use of polyacrylamide gel electrophoresis and isoelectric focusing techniques also suggested that brain AChE exists in multiple forms.     

Monoclonal antibodies against chicken brain acetylcholinesterase. Their use in immunopurification and immunochemistry to demonstrate allelic variants of the enzyme

Acetylcholinesterase (AChE) from 1-day chicken brain was enriched over 2000-fold by affinity chromatography using N-methylacridinium-Sepharose. This preparation was used to prepare monoclonal antibodies (mAb) directed against AChE, of which two were extensively characterised for further application. Both mAbs bound to the enzyme from the chicken with high affinity (Kd approximately 8 X 10(-10) M) and one mAb, in addition, recognised AChE from quail brain and muscle. Neither mAb cross-reacted with mammalian or fish AChE. Both mAbs recognised AChE in the endplate region of adult chicken skeletal muscle and bound with equal affinity to the three major oligomeric forms found in early ambryonic muscle. One mAb was used to immunopurify chicken brain AChE to homogeneity (over 12000-fold enrichment), with nearly complete recovery of the enzyme and without detectable proteolytic breakdown. The other mAb recognised AChE after immunoblotting and was used to screen crude brain extracts from individual chickens for allelic variations. Evidence is presented to show that two allelic forms occur, represented in SDS-PAGE by a doublet polypeptide of Mr approximately 110,000, this pattern is maintained after deglycosylation of the N-linked oligosaccharides. This variation was found throughout development and in both the brain and the muscle of individuals. We conclude that the gene encoding the catalytic subunit of chicken AChE is polymorphic with either one or two equally active alleles being expressed.     

High-level secretion and purification of recombinant acetylcholinesterase from human cerebral tissue in P. pastoris and identification by chromogenic reaction

The gene encoding human cerebral tissue acetylcholinesterase (AChE) was cloned from an 18-week fetal cerebral tissue and expressed in Pichia pastoris. Twenty-two positive transformants were obtained by Mut⁺/Mut^s phenotypes screening in MD/MM medium and polymerase chain reaction amplification, and four recombinant P. pastoris strains that could secrete active AChE at high level were identified by simple and specific development reaction with indoxyl acetate as the chromogenic substrate. In shake-flask culture induced with methanol, the recombinant human AChE (rhAChE) content was about 76% of the total secreted proteins, and rhAChE activity in supernatant was 40 U/ml. The enzyme was purified through anion-exchange and affinity chromatography. Purity of the rhAChE was up to 96% after the simple purification procedure. The enzymatic activity reached 200 U/mg.     

Direct analysis of the kinetic profiles of organophosphate-acetylcholinesterase adducts by MALDI-TOF mass spectrometry

A sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry procedure has been established for the detection and quantitation of acetylcholinesterase (AChE) inhibition by organophosphate (OP) compounds. Tryptic digests of purified recombinant mouse AChE (mAChE) were fractionally inhibited by paraoxon to form diethyl phosphoryl enzyme. The tryptic peptide of mAChE that contains the active center serine residue resolves to a molecular mass of 4331.0 Da. Phosphorylation of the enzyme by paraoxon results in covalent modification of the active center serine and a corresponding increase in molecular mass of the tryptic peptide by 136 Da. The relative abundance of AChE peptides containing a modified active center serine strongly correlates with the fractional inhibition of the enzyme, achieving a detection range of phosphorylated to nonphosphorylated enzyme of 5-95%. Modifications of AChE by OP compounds resulting in dimethyl, diethyl, and diisopropyl phosphoryl adducts have been monitored with subpicomole amounts of enzyme. The individual phosphorylated adducts of AChE that result from loss of one alkyl group from the inhibited enzyme (the aging reaction) and the reappearance of unmodified AChE (spontaneous reactivation) have been resolved by the kinetic profiles and relative abundance of species. Further, the tryptic peptide containing the active center serine of AChE, isolated from mouse brain by anion-exchange and affinity chromatography, has been monitored by mass spectrometry. Native brain AChE, purified from mice treated with sublethal doses of metrifonate, has demonstrated that enzyme modifications resulting from OP exposure can be detected in a single mouse brain. For dimethyl phosphorylated AChE, OP exposure has been monitored by the ratio of tryptic peptide peaks that correspond to unmodified (uninhibited and/or reactivated), inhibited, and aged enzyme.     

Peripheral site ligand-oxime conjugates: A novel concept towards reactivation of nerve agent-inhibited human acetylcholinesterase

A conceptually novel approach to the design of reactivators of nerve agent-inhibited acetylcholinesterase (AChE) is presented. The concept comprises the linkage of a peripheral site ligand via a spacer to a reactivating moiety with the eventual goal to develop non-ionic reactivators with sufficient affinity for AChE to induce reactivation and potentially improved blood-brain barrier penetration. Herein, the first step towards that goal—the synthesis and biological evaluation of a peripheral site ligand conjugated to a charged pyridinium oxime is discussed. It was found, that the introduction of the peripheral site ligand not only increased affinity of the construct for AChE but also enhanced reactivation of nerve agent-inhibited AChE.     

Fast affinity purification coupled with mass spectrometry for identifying organophosphate labeled plasma butyrylcholinesterase

Classical plasma butyrylcholinesterase (BChE) purification involves dialysis and multiple steps of chromatography. We describe a procainamide affinity gel purification scheme that takes 15-30min to purify BChE from 1ml plasma. The method uses a microfuge spin column to build a 0.2ml procainamide affinity column. The eluted BChE contains 3-4mug of 500-fold purified BChE, free from 99% of contaminating plasma proteins. The BChE was further purified by gel electrophoresis. Tryptic peptides from the BChE containing gel electrophoresis band were prepared by in-gel digestion, separated by reverse phase liquid chromatography and identified by mass spectrometry. The 29 residue active site tryptic peptide labeled with the nerve agents soman or sarin was identified.     

Pilot-scale production of human serum butyrylcholinesterase suitable for use as a bioscavenger against nerve agent toxicity

Human serum butyrylcholinesterase (Hu BChE) is currently the most appropriate candidate for the prophylaxis of humans against organophosphate (OP) nerve agent toxicity. It is estimated that a dose of 200 mg will protect a human against 2× LD₅₀ of soman, which means that gram quantities of enzyme are needed for human clinical studies. Toward this effort, we report the development of the first procedure that is suitable for the pilot-scale purification of Hu BChE from Cohn fraction IV-4 paste. This procedure involved resuspension of Cohn fraction IV-4 paste, followed by procainamide affinity and DEAE anion-exchange chromatography. The procedure yielded 6–7 g (4.3–5 million U) of purified enzyme from 80 kg of Cohn fraction IV-4 paste. The enzyme was >97% pure as judged by a specific activity of ∼700 U/mg and a major band with a subunit molecular weight of 85 kDa on SDS-PAGE. The high yield and purity obtained suggest that this manufacturing procedure is suitable for the pre-clinical production of Hu BChE.