Search

Butyrylcholinesterase (BChE) generally acts as an important plasma biomarker for clinical diagnosis due to its major contribution to human plasma cholinesterase levels, but its current fluorometric assay relying on fluorogenic substrates frequently suffers from the lack of sufficiently fast response time and specific recognition of substrates relative to the traditional Ellman's method. In this work, we report a fluorescent molecular probe for assaying BChE activity based on thiol-triggered fluorescence enhancement via thiol-ene click reactions. A low-temperature experiment and theoretical analysis exclude the possibility of weak fluorescence of the probe caused by an intramolecular photoinduced electron transfer process and support the main cause of an ultraslow radiative rate due to the introduction of two acrylyl groups. This probe has sensitive fluorescence responses to thiols via thiol-ene click chemistry, and it can distinguish between glutathione and cysteine or homocysteine in different emission colors. The rapid reaction kinetics of this probe enables it to monitor hydrolysis reactions catalyzed by butyrylcholinesterase (BChE) in a real-time manner. This probe is used to develop the first fluorometric assay of BChE activity based on fluorescence enhancement triggered by thiol-ene click chemistry using butyrylthiocholine as the substrate. The established BChE assay shows excellent sensitivity, and is capable of avoiding the interference from glutathione and acetylcholinesterase (AChE) in a complex matrix. The inhibition test of tacrine on BChE with this assay substantiates its feasibility in screening potential inhibitors of BChE. This work demonstrates a design strategy of fluorescent probes lighted up by thiol-ene click reactions, reveals the main cause of thiol-triggered fluorescence enhancement by altering the radiative rate, and provides the first fluorometric assay of BChE based on rapid thiol-ene click reactions.

In this paper, we, for the first time, describe the interaction between the butyrylcholinesterase enzyme and echothiophate, a popular model compound and an analogue of the chemical warfare agents VX and VR, at the atomistic level. Competition between the two echothiophate conformations in the active site was found using molecular modeling techniques. The first one is close to the mode of binding of the substrates of choline series (butyrylcholine and butyrylthiocholine) and is inhibitory, since it is unable to react with the enzyme. The second one is characterized by a significantly worse estimated binding affinity and is reactive. Thus, echothiophate combines the features of two types of inhibitors: competitive and suicidal. This observation will help clarify the kinetic reaction scheme in order to accurately assess the kinetic constants, which is especially important when designing new butyrylcholinesterase variants capable of full-cycle hydrolysis of organophosphorus compounds.

To identify and characterize specific esterases from S. mutans with degradative activity toward methacrylate-based resin monomers.Out of several putative esterases, an esterase encoded in an Open Reading Frame as SMU_118c (The National Center for Biotechnology Information, NCBI), was found to have true hydrolase activities. SMU_118c was cloned, expressed, purified and further characterized for its respective hydrolytic activity towards ester-containing nitrophenyl substrates and the universal resin monomers bis-phenyl-glycidyl-dimethacrylate (bisGMA) and triethyleneglycol dimethacrylate (TEGDMA) at neutral (7.0) or cariogenic (5.5) pH. Mass spectrometry (MS) was used to verify the expression of SMU_118c protein in S. mutans UA159.Similar to the whole cell activity of S. mutans, SMU_118c showed the highest affinity toward para-nitrophenyl acetate (pNPA) and para-nitrophenyl butyrate (pNPB) vs. ortho-nitrophenyl butyrate (oNPB) and butyrylthiocholine iodide (BTC) (p 0.05). The esterase retained 60% of its activity after 21 days and hydrolyzed bisGMA at a higher rate than TEGDMA at both neutral and cariogenic pH (p 0.001), similarly to the predominant human salivary esterase degradative activity. MS confirmed that SMU_118c is an intracellular protein in S. mutans UA159 and expressed under pathogenic (pH 5.5) growth conditions.The similarity in the activity profile to the whole S. mutans bacterial cell, the stability over time at cariogenic pH, the preference to hydrolyze bisGMA and confirmed expression profile suggest that SMU_118c could be a significant contributor to the whole bacterial degradative activity of S. mutans toward the degradation of resin composites, adhesives and the restoration-tooth interface, potentially accelerating restoration's failure.The current study builds upon our highly-cited previous study by Bourbia et al., (JDR, 2013) that reported on that the cariogenic bacterium, S. mutans has esterase-like activities that enable the bacterium to degrade dental composites and adhesives. The current submission is the first to report on the isolation and characterization of the specific esterase activity (SMU_118c) from S. mutans that is a significant contributor to the whole bacterial degradative activity toward the hydrolysis of dental resins. This activity compromises the restoration-tooth interface, increases interfacial bacterial microleakage (Kermanshahi et al., JDR 2010), potentially contributing to the pathogenesis of recurrent caries around resin composite restorations. This represent a significant contribution to the field of biomaterials and their clinical performance.

We previously reported that recombinant human butyrylcholinesterase (rhBChE) complexed with a series of copolymers of poly- l -lysine (PLL) with grafted (polyethylene) glycol (PEG) (i.e., PLL-g-PEG) showed reduced catalytic activity but relatively similar concentration-dependent inactivation of the organophosphorus inhibitor paraoxon. Herein, we compared the kinetics of catalysis (using butyrylthiocholine as the substrate) and inhibition (using four different inhibitors) of free and copolymer-complexed rhBChE. Using scanning electron microscopy, polyionic complexes of rhBChE with three different PLL-g-PEG copolymers (based on PLL size) appeared as spheroid-shaped particles with relatively similar particle sizes (median diameter = 35 nm). Relatively similar particle sizes were also noted using dynamic light scattering (mean = 26–35 nm). The three copolymer-complexed enzymes exhibited reduced kcat (30–33% reduction), but no significant changes in Km. Inhibitory potency (as reflected by the bimolecular rate constant, ki) was similar among the free and copolymer-complexed enzymes when paraoxon was the inhibitor, whereas statistically significant reductions in ki (16–60%) were noted with the other inhibitors. Sensitivity to inactivation by proteases and heat was also compared. Copolymer-complexed enzymes showed lesser time-dependent inactivation by the proteases trypsin and pronase and by heat compared to the free enzyme. Understanding the unique properties of PLL-g-PEG-BChE complexes may lead to enhanced approaches for use of BChE and other protein bioscavengers.

Cholinesterases (ChE), the enzymes whose primary function is the hydrolysis of choline esters, are widely expressed throughout the nature. Although they have already been found in plants and microorganisms, including ascomycete fungi, this study is the first report of ChE-like activity in fungi of the phylum Basidiomycota. This activity was detected in almost a quarter of the 45 tested aqueous fungal extracts. The ability of these extracts to hydrolyse acetylthiocholine was about ten times stronger than the hydrolytic activity towards butyrylthiocholine and propionylthiocholine. In-gel detection of ChE-like activity with acetylthiocholine indicated a great variability in the characteristics of these enzymes which are not characterized as vertebrate-like based on (i) differences in inhibition by excess substrate, (ii) susceptibility to different vertebrate acetylcholinesterase and butyrylcholinesterase inhibitors, and (iii) a lack of orthologs using phylogenetic analysis. Limited inhibition by single inhibitors and multiple activity bands using in-gel detection indicate the presence of several ChE-like enzymes in these aqueous extracts. We also observed inhibitory activity of the same aqueous mushroom extracts against insect acetylcholinesterase in 10 of the 45 samples tested; activity was independent of the presence of ChE-like activity in extracts. Both ChE-like activities with different substrates and the ability of extracts to inhibit insect acetylcholinesterase were not restricted to any fungal family but were rather present across all included Basidiomycota families. This study can serve as a platform for further research regarding ChE activity in mushrooms.

The purpose of this study was to measure and characterize the enzyme cholinesterase (ChE) in porcine saliva, as well as to evaluate its behavior in experimental stressful conditions. The results of ChE characterization by using different substrates and the selective inhibitors ethopropazine and physostigmine showed that the main enzyme existing in porcine saliva was butyrylcholinesterase (BChE). An automated assay using butyrylthiocholine iodide as substrate was validated providing adequate reproducibility, linearity results and limit of detection. Salivary ChE was measured using the validated assay in two models of acute stress: twenty pigs stressed for 2min with a nasal snare and other twenty pigs subjected to a short-term road transport. Salivary ChE significantly increased after restraint and transport stress in pigs, as well as the ChE to total protein ratio. In conclusion, BChE is the predominant isoenzyme in porcine saliva, it can be measured by the fast, simple and automated method described in this paper and it increases in the models of stress used in this study.

A modified enzyme method is reported for the determination of cholinesterase inhibitors based on the use of a granulated composite carrier with immobilized butyrylcholinesterase. The detection system includes butyrylthiocholine as the substrate and N-(2,3-dimethyl-5-oxo-1-phenyl-3-pyrazoline-4-yl)-2-chloro-5-sulpho-4-iminobenzoquinone as a redox indicator. This method was characterized using carbamate physostigmine and sarin (O-isopropyl methylphosphonofluoridate). The method was also used in a simple tube detector. Evaluation was based on the observation with the naked eye of the decolorization of the redox indicator in comparison to a control time and by tristimulus colorimetry. The limits of detection were 0.5 µg/mL for physostigmine and 7 × 10−4 mg/m3 for sarin visually, or 0.06 µg/mL for physostigmine and 1.1 × 10−4 mg/m3 for sarin by colorimetry.

Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger that protects from the toxicity of nerve agents. Non-human primates are suitable models for toxicity studies that cannot be performed in humans. We evaluated the biochemical properties of native macaque (MaBChE) tetramers, compared to recombinant MaBChE monomers, PEGylated recombinant MaBChE tetramers and monomers, and native HuBChE tetramers. Km and kcat values for butyrylthiocholine were independent of subunit assembly status. The Km for all forms of MaBChE was about 70 μM, compared to 13 μM for HuBChE. The kcat was about 100,000 min−1 for MaBChE and 30,000 min−1 for HuBChE. The reversible inhibitor ethopropazine had similar Ki values of 0.05 μM for all MaBChE forms and HuBChE. The bimolecular rate constant, ki, for inhibition by diisopropylfluorophosphate (DFP), an analog of sarin, was 2.2 to 2.5 × 107 M−1 min−1 for all MaBChE forms and for HuBChE. A major difference between MaBChE and HuBChE was the rate of reactivation by 2-PAM. The second order rate constant for reactivation of DFP-inhibited MaBChE by 2-PAM was 1.4 M−1 min−1, but was 380 fold faster for DFP-inhibited HuBChE (kr 531 M−1 min−1). The acyl pocket of MaBChE has Leu285 in place of Pro285 in HuBChE. The reactivation rate of DFP-inhibited HuBChE mutant P285L by 2-PAM was reduced 5.8-fold (kr 92 M−1 min−1) indicating that P285 determines whether 2-PAM binds in an orientation that favors release of diisopropylphosphate. DFP-inhibited MaBChE treated with 0.2 M 2-PAM recovered 10% of its original activity, whereas DFP-inhibited HuBChE recovered 80% activity. It was concluded that the biochemical properties of MaBChE are similar to those of HuBChE except for the reactivation of DFP-inhibited BChE.

A new spectrofluorimetric method more sensitive than the Ellman method was developed for determination of both acetylcholinesterase and butyrylcholinesterase activity and for kinetic analysis of these enzymes and their mutants. Two selected mutants of human butyrylcholinesterase (E197Q and E197G) were included in this work. As for the Ellman's method, substrates are thiocholine esters, but the chromogenic reagent, DTNB (dithio-bisnitro benzoic acid) is replaced by a fluorogenic probe, “Calbiochem Probe IV”, (3-(7-Hydroxy-2-oxo-2H-chromen-3-ylcarbamoyl)acrylic acid methylester). Compared to the classical Ellman's method, the sensitivity of this new spectrofluorimetric assay is 2 orders of magnitude higher. The method allows measurement of activity in media containing

Mouse butyrylcholinesterase (mBChE) and an mBChE-based cocaine hydrolase (mCocH, i.e. the A199S/S227A/S287G/A328W/Y332G mutant) have been characterized for their catalytic activities against cocaine, i.e. naturally occurring (−)-cocaine, in comparison with the corresponding human BChE (hBChE) and an hBChE-based cocaine hydrolase (hCocH, i.e. the A199S/F227A/S287G/A328W/Y332G mutant). It has been demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against (−)-cocaine by ~8- and ~2000-fold respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. According to the kinetic data, the catalytic efficiency ( k cat /K M) of mBChE against (−)-cocaine is comparable with that of hBChE, but the catalytic efficiency of mCocH against (−)-cocaine is remarkably lower than that of hCocH by ~250-fold. The remarkable difference in the catalytic activity between mCocH and hCocH is consistent with the difference between the enzyme–(−)-cocaine binding modes obtained from molecular modelling. Further, both mBChE and hBChE demonstrated substrate activation for all of the examined substrates [(−)-cocaine, ACh and BTC] at high concentrations, whereas both mCocH and hCocH showed substrate inhibition for all three substrates at high concentrations. The amino-acid mutations have remarkably converted substrate activation of the enzymes into substrate inhibition, implying that the rate-determining step of the reaction in mCocH and hCocH might be different from that in mBChE and hBChE. Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; BChE, butyrylcholinesterase; BTC, butyrylthiocholine; CHO, Chinese hamster ovary; CIP, calf intestinal alkaline phospahtase; CocH, cocaine hydrolases; hBChE, human butyrylcholinesterase; hCocH, human-butyrylcholinesterase-based cocaine hydrolase; mBChE, mouse butyrylcholinesterase; mCocH, mouse butyrylcholinesterase-based cocaine hydrolase; MD, molecular dynamics; TS, transition state

Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivarcurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of mivacurium leading to the discovery of a novel BCHE variant (c.185C>T, p.Ala34Val). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation remote from the active center determines the “silent” phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with a heterozygous enzyme of type atypical/silent. Kinetic analysis with succinyldithiocholine (SCdTC) as the substrate showed that Ala34Val BChE was inactive against this substrate. However, with BTC, the mutant enzyme was active, displaying an unexpected activation by excess substrate. Competitive inhibition of BTC by mivacurium gave a K i = 1.35 mM consistent with the lack of activity with the related substrate SCdTC, and with the clinical data. Molecular dynamic simulations revealed the mechanism by which mutation Ala34Val determines the silent phenotype: a chain of intramolecular events leads to disruption of the catalytic triad, so that His438 no longer interacts with Ser198, but instead forms hydrogen bonds either with residues Glu197 and Trp82, or peripheral site residue Tyr332. However, at high BTC concentration, initial binding of substrate to the peripheral site triggers restoration of a functional catalytic triad, and activity with BTC.

Human butyrylcholinesterase (hBChE) is currently being developed as a detoxication enzyme for stoichiometric binding and/or catalytic hydrolysis of organophosphates. Herein, we describe the use of a molecular evolution method to develop novel hBChE variants with increased resistance to stereochemically defined nerve agent model compounds of soman, sarin, and cyclosarin. Novel hBChE variants (Y332S, D340H, and Y332S/D340H) were identified with an increased resistance to nerve agent model compounds that retained robust intrinsic catalytic efficiency. Molecular dynamics simulations of these variants revealed insights into the mechanism by which these structural changes conferred nerve agent model compound resistance.

Organophosphate insecticides (OPs) continue to be an important class of agrochemicals used in modern agriculture worldwide. Even though these pesticides persist in the environment for a relatively short time, they show a high acute toxicity that may represent a serious hazard for wildlife. Sub-lethal effects on non-target species are a focus in pest management programs and should be used as biomarkers. Cholinesterases (ChEs) are the most used biomarker of OP exposure in vertebrate and invertebrate species. However, the combined monitoring of ChE and carboxylesterase (CE) activities may provide a more useful indication of exposure and effect of the organisms. The objective of the present work was to find the most sensitive combination of enzyme, substrate, tissue and capacity to recovery of B-esterases in the freshwater gastropod Planorbarius corneus exposed to the OP azinphos-methyl. For this purpose, ChE and CE activities in different tissues of P. corneus (head-foot, pulmonary region, digestive gland, gonads and whole organism soft tissue) were studied. Measurements of ChE activity were performed using three substrates: acetylthiocholine, propionylthiocholine and butyrylthiocholine and CE activity using four different substrates: p-nitrophenyl acetate, p-nitrophenyl butyrate, 1-naphthyl acetate, and 2-naphthyl acetate in control and exposed organisms. Finally, the recovery rates of ChE and CE activities following 48 h exposure to azinphos-methyl were analyzed. Our results show a preference for acetylthiocholine as substrate, a high inhibition with eserine (a selective ChE inhibitor) and inhibition with excess of substrate in all the analyzed tissues. The highest ChE and CE activity was found in the pulmonary region and in the digestive gland, respectively. The highest CE Vmax was obtained with 1 and 2-naphthyl acetate in all the tissues. CEs were more sensitive than ChE to azinphos-methyl exposure. The highest sensitivity was found using p-nitrophenyl acetate and butyrate as substrates. On the other hand, CEs of the digestive gland and the pulmonary region were more sensitive than CEs of the whole organism soft tissue. Regarding the recovery of enzyme activities after 48 h exposure, ChE and CEs with p-nitrophenyl butyrate reached control values after 14 days in the digestive gland and after 21 days in the pulmonary region. Our results show marked differences in P. corneus basal ChE and CE activities depending on substrates and the tissue. Also, both tissue-dependent and substrate-dependent variations in sensitivity to azinphos-methyl exposure and recovery were obtained. CEs measured with p-nitrophenyl butyrate in the pulmonary region were the best combination to be used as biomarker of exposure to azinphos-methyl due to their sensitivity and low recovery capacity. Environmental concentrations of azinphos-methyl inhibited CE activity so they could be used as effective biomarkers of aquatic contamination. Fil: Otero, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Kristoff, Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

In this study, the cholinesterase (ChE) and carboxylesterase (CES) activities present in whole organism homogenates from Planorbarius corneus and their in vitro sensitivity to organophosphorous (OP) pesticides were studied. Firstly, a characterization of ChE and CES activities using different substrates and selective inhibitors was performed. Secondly, the effects of azinphos-methyl oxon (AZM-oxon) and chlorpyrifos oxon (CPF-oxon), the active oxygen analogs of the OP insecticides AZM and CPF, on ChE and CES activities were evaluated. Finally, it was analyzed whether binary mixtures of the pesticide oxons cause additive, antagonistic or synergistic ChE inhibition in P. corneus homogenates. The results showed that the extracts of P. corneus preferentially hydrolyzed acetylthiocholine (AcSCh) over propionylthiocholine (PrSCh) and butyrylthiocholine (BuSCh). Besides, AcSCh hydrolyzing activity was inhibited by low concentrations of BW284c51, a selective inhibitor of AChE activity, and also by high concentrations of substrate. These facts suggest the presence of a typical AChE activity in this species. However, the different dose-response curves observed with BW284c51 when using PrSCh or BuSCh instead of AcSCh suggest the presence of at least another ChE activity. This would probably correspond to an atypical BuChE. Regarding CES activity, the highest specific activity was obtained when using 2-naphthyl acetate (2-NA), followed by 1-naphthyl acetate (1-NA); p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). The comparison of the IC50 values revealed that, regardless of the substrate used, CES activity was approximately one order of magnitude more sensitive to AZM-oxon than ChE activity. Although ChE activity was very sensitive to CPF-oxon, CES activity measured with 1-NA, 2-NA, and p-NPA was poorly inhibited by this pesticide. In contrast, CES activity measured with p-NPB was equally sensitive to CPF-oxon than ChE activity. Several specific binary combinations of AZM-oxon and CPF-oxon caused a synergistic effect on the ChE inhibition in P. corneus homogenates. The degree of synergism tended to increase as the ratio of AZM-oxon to CPF-oxon decreased. These results suggest that synergism is likely to occur in P. corneus snails exposed in vivo to binary mixtures of the OPs AZM and CPF. Fil: Cacciatore, Luis Claudio. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Kristoff, Gisela. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Verrengia Guerrero, Noemí Rosario. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Cochon, Adriana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Cholinesterases and carboxylesterases belong to the group of B-esterases, the serine superfamily of esterases that are inhibited by organophosphorus compounds. It is now generally accepted that before using the B-esterases as biomarkers of exposure to organophosphorus and carbamates in a given species, the biochemical characteristics of these enzymes should be carefully studied. In this study, the enzyme/s and the tissue/s to be selected as sensitive biomarkers of organophosphorus exposition in the freshwater gastropod Biomphalaria glabrata were investigated. Firstly, the substrate dependence of cholinesterase and carboxylesterase activities in whole organism soft tissue and in different tissues of the snail (head-foot, pulmonary region, digestive gland, and gonads) was analyzed. Measurements of cholinesterase activity were performed using three substrates: acetylthiocholine (AcSCh), propionylthiocholine (PrSCh), and butyrylthiocholine (BuSCh). Carboxylesterase activity was determined using four different substrates: 1-naphthyl acetate (1-NA), 2-naphthyl acetate (2-NA), p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). Regardless of the tissue analyzed, the highest specific activity was obtained when using AcSCh, followed by PrSCh. Cholinesterase activity measured with BuSCh was very low in all cases. On the other hand, the highest cholinesterase activity was measured in head-foot and in pulmonary region, representing in the case of AcSCh hydrolysis 196% and 180% of the activity measured in whole organism soft tissue, respectively. In contrast, AcSCh hydrolysis in digestive gland and gonads was 28% and 50% of that measured in whole organism soft tissue. Regarding carboxylesterase activity, although all tissues hydrolyzed the four substrates assayed, substrate preferences varied among tissues. In particular, digestive glands showed higher carboxylesterase activity than the other tissues (299%, 359% and 137% of whole organism soft tissue activity) when measured with 1-NA, 2-NA and p-NPA as substrates, respectively. In contrast, with p-NPB as substrate, the highest carboxylesterase activity was observed in pulmonary region. Exposure of the snails for 48 h to azinphos-methyl concentrations in the range of 0.05-2.5 mg L⁻¹ resulted in different degrees of inhibition of cholinesterase and carboxylesterase activities, depending on the enzyme, pesticide concentration, the substrate, and the tissue analyzed. In general, carboxylesterase activity measured with p-NPA and p-NPB was much more sensitive to azinphos-methyl inhibition than cholinesterase activity. The results also showed that while B-esterase activities in whole organism soft tissue and pulmonary region recovered completely within 14 days, carboxylesterase activity in digestive glands remained highly inhibited. On the whole, the results of the present study emphasize how important it is to characterize and measure cholinesterase and carboxylesterase activities jointly to make a proper assessment of the impact of organophosphorus pesticides in non-target species.

Cationic triarylmethane dyes (TAM(+))s which are used as colorants in industry and as frequent tools and reagents in analytical, cell biological and biomedical research have been recently characterized as reversible inhibitors of human butyrylcholinesterase. In this study, the inhibitory effects of two TAM(+)s, malachite green (MG) and methyl green (MeG) on five human BChE mutants (A277V, P285L, H77L, A328F and F329A) were studied spectrophotometrically at 25 degrees C in 50 mM MOPS buffer pH 8, using butyrylthiocholine as substrate. The kinetic results obtained with mutant enzymes were compared to those obtained with recombinant wild type BChE. MG and MeG were found to act as competitive/linear mixed inhibitors of recombinant wild type BChE and all BChE mutants except the F329A mutant. Both dyes caused complex nonlinear inhibition of F329A mutant, pointing to multisite binding. K-i values for MG and MeG, estimated by nonlinear regression analysis, were 3.8 and 27 mu M, respectively, as compared to the 50- to 150-fold lower values observed with recombinant wild type BChE. The observed significant differences in kinetic pattern and K-i values between recombinant wild type BChE and F329A mutant suggest that phenylalanine at position 329 in human BChE is a critical residue in MG and MeG binding to enzyme. (C) 2011 Elsevier Inc. All rights reserved.

Triatoma patagonica Del Ponte (Hemiptera: Reduviidae), a vector of Chagas' disease, is widely distributed in Argentina and is found in sylvatic and peridomiciliary ecotopes, as well as occasionally in human dwellings after the chemical control of Triatoma infestans. Anti-cholinesteratic products can be applied in peridomiciliary areas and thus knowledge of cholinesterase activity during embryonic development in this species might contribute further information relevant to effective chemical control. Cholinesterase activity was characterized by reactions to eserine 10 -5m, to increasing concentrations of substrate and to varying centrifugal speeds. Acetylcholinesterase activity was detected on day 4 and was significant from day 5. A reduction in cholinesterase activity towards acetylthiocholine (ATC) was observed on days 9 and 10 of development. Cholinesterase activity towards ATC and butyrylthiocholine (BTC) in homogenates of eggs was inhibited by eserine 10 -5m. The shape of the curve indicating levels of inhibition at different concentrations of ATC was typical of acetylcholinesterase. Activity towards BTC did not appear to be inhibited by excess substrate, which parallels the behaviour of butyrylcholinesterases. Cholinesterase activity towards ATC was reduced in supernatant centrifuged at 15 000 g compared with supernatant centrifuged at 1100 g. The cholinesterase system that hydrolyzes mainly ATC seems to belong to the nervous system, as indicated by its behaviour towards the substrates assayed, its greater insolubility and the fact that it evolves parallel to the development of the nervous system. Knowledge of biochemical changes associated with the development and maturation of the nervous system during embryonic development would contribute to the better understanding of anti-cholinesteratic compounds with ovicidal action that might be used in control campaigns against vectors of Chagas' disease. Fil: Visciarelli, Elena Concepcion. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Chopa, Alicia Beatriz. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Picollo, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Ferrero, Adriana Alicia. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina

Human serum Butyrylcholinesterase (BChE) is an important enzyme in detoxification with its capacity for hydrolyzing esters. The inhibitory effect of cisplatin (CDDP) and cyclophosphamide (CY) on BChE is characterized. Time dependent inhibition of BChE with both chemotherapeutics was rapid, reversible. CY was found as non-competitive inhibitor with Ki of 503.6 +/- 50.4 microM. Time dependent CDDP studies displayed progressive inhibition. The constants for apparent dissociation (Ka), first order constant for the break down of the Michaelis complex (k + 2), and bimolecular rate (ka) were calculated as 6.38 x 10(-5) M(-1) min(-1), 0.063 min(-1), and 9.83 x 10(-4) M, respectively. Enzyme protection could be achieved with moderate butyrylthiocholine concentrations (0.3 mM) but higher concentrations increased CDDP inhibition. Apparent Ki value for CDDP was 191.8 +/- 71.2 microM. These results suggest that used in combination therapy, CY and CDDP cause considerable BChE inhibition and may aggravate conditions observed during chemotherapy.

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) are enzymes that belong to the superfamily of α/β-hydrolase fold proteins. While they share many characteristics, they also possess many important differences. For example, whereas they have about 54% amino acid sequence identity, the active site gorge of acetylcholinesterase is considerably smaller than that of butyrylcholinesterase. Moreover, both have been shown to display simple and complex kinetic mechanisms, depending on the particular substrate examined, the substrate concentration, and incubation conditions. In the current study, incubation of butyrylthiocholine in a concentration range of 0.005–3.0 mM, with 317 pM human butyrylcholinesterase in vitro, resulted in rates of production of thiocholine that were accurately described by simple Michaelis–Menten kinetics, with a K m of 0.10 mM. Similarly, the inhibition of butyrylcholinesterase in vitro by the organophosphate chlorpyrifos oxon was described by simple Michaelis–Menten kinetics, with a k i of 3048 nM −1 h −1 , and a K D of 2.02 nM. In contrast to inhibition of butyrylcholinesterase, inhibition of human acetylcholinesterase by chlorpyrifos oxon in vitro followed concentration-dependent inhibition kinetics, with the k i increasing as the inhibitor concentration decreased. Chlorpyrifos oxon concentrations of 10 and 0.3 nM gave k i s of 1.2 and 19.3 nM −1 h −1 , respectively. Although the mechanism of concentration-dependent inhibition kinetics is not known, the much smaller, more restrictive active site gorge of acetylcholinesterase almost certainly plays a role. Similarly, the much larger active site gorge of butyrylcholinesterase likely contributes to its much greater reactivity towards chlorpyrifos oxon, compared to acetylcholinesterase.

The kinetic effects of a selection of triarylmethane, phenoxazine and phenothiazine dyes (pararosaniline (PR), malachite green (MG), methyl green (MeG); meldola blue (MB), nile blue (NB), nile red (NR); methylene blue (MethB)) and of ethopropazine on horse serum butyrylcholinesterase were studied spectrophotometrically at 25 degrees C in 50 mM MOPS buffer, pH 8, using butyrylthiocholine as substrate. PR, MeG, MB and ethopropazine acted as linear mixed type inhibitors of the enzyme, with respective K(i) values of 4.5 +/- 0.50 mu M, 0.41 +/- 0.007 mu M, 0.44 +/- 0.086 mu M and 0.050 +/- 0.0074 mu M. MG, NB, MethB and NR caused complex, nonlinear inhibition pointing to cooperative binding at two sites. Intrinsic K ' values ([I](2)(0.5) extrapolated to [S]=0) for MG, NB, NR and MethB were 0.20 +/- 0.096 mu M, 0.0018 +/- 0.0015 mu M, 0.92 +/- 0.23 mu M and 0.23 +/- 0.08 mu M. NB stood out as a potent inhibitor effective at nM levels. Comparison of inhibitory effects on horse and human serum butyrylcholinesterases suggested that the two enzymes must have distinct microstructural features. (c) 2008 Elsevier Inc. All rights reserved.

Human butyrylcholinesterase (hBChE) is a highly glycosylated protein present in human plasma. The enzyme hydrolyses choline esters, for example benzoylcholine, butyrylthiocholine and acetylthiocholine as well as noncholine esters like heroin and aspirin. hBChE is primarily involved in neuronal transmission and is a potential bioscavenger of toxic organophosphates to protect acetylcholinesterase. A prerequisite for the therapeutic use of hBChE is a detailed characterization of this glycoprotein purified from human plasma. In this study, MS/MS could confirm most of the protein backbone, including the N- and the C-terminus. Site-specific analysis of all nine potential N-glycosylation sites revealed mainly mono- and disialylated N-glycans to be present on this glycoprotein. Sialic acids (Neu5Ac) are mainly alpha2,6-linked, however a fraction of the N-glycans contained Neu5Ac also in alpha2,3 linkage. On monosialylated N-glycans, sialic acid is exclusively located on the 3-arm and in alpha2,6 linkage, as verified by 2D-HPLC and exoglycosidase digests of 2-aminopyridine (PA)-labelled N-glycans. This first comprehensive glycoproteomic analysis of the important human plasma glycoprotein BChE did not give any indication of O-glycosylation or any other kind of PTMs as previously postulated.

We recently identified plant acetylcholinesterases (E.C.3.1.1.7; AChEs) homologous to the AChE purified from a monocotyledon, maize, that are distinct from the animal AChE family. In this study, we purified, cloned and characterized an AChE from a dicotyledon, siratro. The full-length cDNA of siratro AChE is 1,441 nucleotides, encoding a 382-residue protein that includes a signal peptide. This AChE is a disulfide-linked 125-kDa homotrimer consisting of 41-42 kDa subunits, in contrast to the maize AChE, which exists as a mixture of disulfide and non-covalently linked 88-kDa homodimers. The plant AChEs apparently consist of various quaternary structures, depending on the plant species, similar to the animal AChEs. We compared the enzymatic properties of the dimeric maize and trimeric siratro AChEs. Similar to electric eel AChE, both plant AChEs hydrolyzed acetylthiocholine (or acetylcholine) and propionylthiocholine (or propionylcholine), but not butyrylthiocholine (or butyrylcholine), and their specificity constant was highest against acetylcholine. There was no significant difference between the enzymatic properties of trimeric and dimeric AChEs, although two plant AChEs had low substrate turnover numbers compared with electric eel AChE. The two plant AChE activities were not inhibited by excess substrate concentrations. Thus, similar to some plant AChEs, siratro and maize AChEs showed enzymatic properties of both animal AChE and animal BChE. On the other hand, both siratro and maize AChEs exhibited low sensitivity to the AChE-specific inhibitor neostigmine bromide, dissimilar to other plant AChEs. These differences in enzymatic properties of plant AChEs may reflect the phylogenetic evolution of AChEs.

Cholinergic loss is the single most replicated neurotransmitter deficiency in Alzheimer's disease (AD) and has led to the use of acetylcholinesterase inhibitors (AChE-Is) and unselective cholinesterase inhibitors (ChE-Is) as the mainstay of treatment. AChE-Is and ChE-Is, however, induce dose-limiting adverse effects. Recent studies indicate that selective butyrylcholinesterase inhibitors (BuChE-Is) elevate acetylcholine (ACh) in brain, augment long-term potentiation, and improve cognitive performance in rodents without the classic adverse actions of AChE-Is and ChE-Is. BuChE-Is thereby represent a new strategy to ameliorate AD, particularly since AChE activity is depleted in AD brain, in line with ACh levels, whereas BuChE activity is elevated. Our studies have focused on the design and development of cymserine analogues to induce selective time-dependent brain BuChE inhibition, and on the application of innovative and quantitative enzyme kinetic analyses to aid selection of drug candidates. The quantitative interaction of the novel inhibitor, dihydrobenzodioxepine cymserine (DHBDC), with human BuChE was characterized. DHBDC demonstrated potent concentration-dependent binding with BuChE. The IC(50) and specific new kinetic constants, such as K(T50), P(PC), K(T1/2) and R(I), were determined at dual substrate concentrations of 0.10 and 0.60 mM butyrylthiocholine and reaction times, and are likely attainable in humans. Other classical kinetic parameters such as K(ia), K(ma), V(ma) and V(mi) were also determined. In synopsis, DHBDC proved to be a highly potent competitive inhibitor of human BuChE in comparison to its structural analogue, cymserine, and represents an interesting drug candidate for AD.

Over the last decades, cholinesterase (ChE) biosensors have emerged as a sensitive and rapid technique for toxicity analysis in environmental monitoring, food, and quality control. These systems have the potential to complement or replace the classical analytical methods by simplifying or eliminating sample preparation protocols and making field testing easier and faster with significant decrease in costs per analysis. In this study, a new bienzymatic biosensor based on butyrylcholinesterase (BuChE) and sulfhydryl oxidase (SOX) enzymes was developed. This system makes use of the biocatalyzed hydrolysis of butyrylthiocholine to butyric acid and thiocholine that acts as a SOX substrate. Measurements were performed by following of the consumed oxygen level related to butyrylthiocholine concentration in the enzymatic reactions. Bienzymatic system was characterized and applied for detection of donepezil which is a reversible inhibitor of cholinesterase and belongs to a new class of cholinesterase inhi...

Cholinesterases (ChEs) have been characterized in marbled sole (Limanda yokohamae) for use as a possible biomarker of pollution exposure. In brain, ChEs existed almost exclusively (>95%) as acetylcholinesterase (AChE) whereas in muscle, about 20-30% of ChE activity was in the form of butyrylcholinesterase (BChE; pseudocholinesterase). Acetylthiocholine and butyrylthiocholine (identified in mammalian studies as diagnostic substrates for AChE and BChE respectively) were hydrolyzed mainly, but not exclusively, by these enzymes. The inhibitors BW284C51 and iso-OMPA (identified in mammalian studies as diagnostic inhibitors of AChE and BChE respectively) were not specific for these enzymes in marbled sole. Brain AChE and muscle AChE and BChE were characterized in terms of their kinetic properties (KM etc.) and optimal conditions (substrate concentration, protein concentration, pH etc.) were established to allow routine assays of ChE activity to proceed under pseudo-first order conditions. The sensitivity of ChEs to a locally significant pesticide, iprobenfos (IBP; kitazin) was established in terms of IC50 concentrations. Brain AChE was relatively insensitive to IBP, but muscle AChE and BChE were sensitive to IBP concentrations in the high nM range. However, ambient IBP concentrations in Korean coastal waters are usually not high enough to cause detectable ChE inhibition in this species.

Two cationic phenoxazine dyes, meldola blue (MB) and nile blue (NB), and the structurally related phenothiazine, methylene blue (MethB), were found to act as complex inhibitors of human plasma cholinesterase (butyrylcholinesterase, BChE). Studied at 25 °C, in 100 mM MOPS buffer (pH 8.0), with butyrylthiocholine as substrate, the kinetic pattern of inhibition indicated cooperative I binding at 2 sites. Intrinsic K′ values ( ≡ [ I ] 0.5 2 extrapolated to [S] = 0) for MB, NB and MethB were 0.64 ± 0.05, 0.085 ± 0.026 and 0.42 ± 0.04 μM, respectively. Under the same experimental conditions the dyes acted as single-occupancy, hyperbolic-mixed inhibitors of electric eel acetylcholinesterase (AChE), with Ki = 0.035 ± 0.010, 0.026 ± 0.0034 and 0.017 ± 0.0063 μM (for MB, NB, MethB); α (coefficient of competitive interaction) = 1.8–2.4 and β (coefficient of noncompetitive interaction) = 0.15–0.28. The complexity of the BChE inhibitory effect of phenoxazine/phenothiazine dyes contrasted with that of conventional ChE inhibitors which cause single-occupancy (n = 1), competitive or mixed inhibition in both AChE and BChE and signaled novel modes of ligand interaction at (or remote from) the active site gorge of the latter enzyme.

International audience; Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18e5, B2 12e1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18e5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18e5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18e5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18e5 and mAb2 can be used to immuno extract BChE from the plasma of humans, monkey and other animals.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

Assessment of pollution impact in soil ecosystems has become a priority and interest has grown concerning the use of invertebrates as sentinel organisms. Inhibition of cholinesterase (ChE) activity has a great potential as a biomarker of pesticide exposure, and we evaluated the ChE kinetic parameters in the earthworm Eisenia andrei in the presence of acetylthiocholine (ASCh), proprionylthiocholine (PSCh) and butyrylthiocholine (BSCh). The highest ChE activity was found in the presence of ASCh and PSCh (42.45 and 49.82 nmol min− 1 mg protein− 1, respectively). BSCh was hydrolyzed at a rate of 4.04 nmol min− 1 mg protein− 1, but the time course did not reach a plateau under our experimental conditions. Km values were 0.142 ± 0.006 and 0.183 ± 0.053 mM for ASCh and PSCh, respectively. ASCh and PSCh hydrolysis were significantly inhibited by eserine (IC50 values were 1.44 × 10− 8 and 1.20 × 10− 8 M, respectively) and by carbaryl (IC50 values of 5.75 × 10− 9 and 4.79 × 10− 9 M). The presence of different ChEs in tissues from E. andrei was assessed by using selective inhibitors for AChE (BW284c51) and BChE (iso-OMPA). BW284c51 strongly reduced ASCh and PSCh hydrolysis and slightly affected that of BSCh, while iso-OMPA was without effect in all cases.

Butyrylcholinesterase (BChE) constitutes the first line defense in the serum of higher organisms and is a marker for toxic exposure. Indole-3-acetic acid (IAA) is a major plant growth hormone of the auxin class, affecting cell enlargement, and differentiation. As a result of the industrial usage, this agrochemical is consumed by non-target organisms. In this study, the interaction of IAA with purified human and horse serum BChEs were investigated. Both BChE species displayed biphasic hill plots using butyrylthiocholine as substrate. IAA interaction with BChE species was stable and concentration dependent. IAA was found to be linear-mixed type inhibitor for human serum BChE, and alpha and the Ki values were 2.15 +/- 1.09 mM and 3.09 +/- 0.95 mM, respectively. With horse enzyme IAA displayed uncompetitive inhibition with the Ki value of 1.05 +/- 0.09 mM. Acquisition of large amounts of IAA is unlikely but it can be taken as a basis for future inhibitor designs.

Cholinesterases (ChE) from brain, muscle and liver in Nile tilapia (Oreochromis niloticus) were characterized using three substrates: acetylthiocholine iodide, propionylthiocholine iodide, and butyrylthiocholine iodide. Eserine was used as a total ChE inhibitor; BW284c51 and iso-OMPA were used as selective inhibitors for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), respectively. The results indicate that AChE is the enzyme present in brain, whereas in both liver and muscle, the presence of atypical ChEs are suggested. These findings indicate that characterization of ChE is necessary prior to use in monitoring programs.

A hydrophilic form of acetylcholinesterase (AChE) was purified from N-methyl carbamate susceptible (SA) and highly N-methyl carbamate-resistant (N3D) strains of the green rice leafhopper (GRLH), Nephotettix cincticeps Uhler. Both of purified AChE from SA and N3D strains displayed the highest activities toward acetylthiocholine (ATCh) at pH 8.5. In the SA strain, the optimum concentrations for ATCh, propionylthiocholine (PTCh), and butyrylthiocholine (BTCh) were about 1 × 10−3, 2.5 × 10−3, and 1 × 10−3 M, respectively. However, in the N3D strain, substrate inhibition was not identified for ATCh, PTCh, and BTCh to 1 × 10−2 M. The Km value in the SA strain was 51.1, 39.1, and 41.6 μM and that in the N3D strain was 91.8, 88.1, and 85.2 μM for ATCh, PTCh, and BTCh, respectively. The Km value in the N3D strain indicated about 1.80-, 2.25-, and 2.05-fold lower affinity than that of the SA strain for ATCh, PTCh, and BTCh, respectively. The Vmax value in the SA strain was 70.2, 30.5, and 4.6 U/mg protein and that in the N3D strain was 123.0, 27.0, and 14.5 U/mg protein for ATCh, PTCh, and BTCh, respectively. The Vmax value in the N3D strain was 1.75- and 3.15-fold higher for ATCh and BTCh than that in the N3D strain. However, it was 1.13-fold lower for PTCh. The increased activity of AChE in the N3D strain is due to the qualitatively modified enzyme with a higher catalytic efficiency. The bimolecular rate constant (ki) for propoxur was 27.1 × 104 and 0.51 × 104 M−1 min−1 in the SA and N3D strain and that for monocrotophos was 0.031 × 104 and 2.0 × 104 M−1 min−1 in the SA and N3D strain. AChE from the N3D strain was 53-fold less sensitive than SA strain to inhibition by propoxur. In contrast, AChE from the N3D strain was 65-fold more sensitive to inhibition by monocrotophos than AChE from the SA strain. This indicated negatively correlated cross-insensitivity of AChE to propoxur and monocrotophos.

Human butyrylcholinesterase (HuBChE) is of particular interest because it hydrolyzes a wide range of toxic compounds such as cocaine, succinylcholine and carbamate pesticides. In this study, HuBChE was purified from human plasma using polyethylene glycol (PEG), differential precipitation and ion exchange chromatographic procedures. Purification resulted in a homogeneous enzyme preparation with a 5575-fold purification, and specific activity of 485 U/mg proteins with a reasonable final yield (36%). The final purified HuBChE showed a single electrophoresis band (85 kDa) on SDS-PAGE. Purified enzyme was stabilized with glycerol, PEG, and ammonium sulphate. Kinetic studies of the enzyme showed high affinity for butyrylthiocholine ( k m =18±5 μM) and low affinity for acetylthiocholine ( k m =83±12 μM), paraoxon, parathion, and tetra monoisopropyl pyrophosphomide all inhibited the enzyme with IC 50 of 2.8±0.8, 22±2 and 55±4 μM, respectively. The procedure introduced is inexpensive, simple and comparable to procainamide affinity column chromatography. This procedure can provide large quantities of purified enzyme with acceptable stability.

Butyrylcholinesterase (BChE; E.C. 3.1.1.8.) was 260-fold purified from soluble fraction of rat intestine. The enzyme was composed of tetrameric globular form by nonreducing electrophoresis. Optimum pH value was determined as 7.2 after zero buffer extrapolation. Optimum temperature was examined as 37 degrees C after zero time extrapolation. The enzyme showed marked substrate activation with positively charged, acyl-choline substrates. As a measure of catalytic efficiency, kcat/Km values were determined as 16,210, 25,650, and 46,150 for acetylthiocholine (ATCh), propionylthiocholine (PTCh), and butyrylthiocholine (BTCh), respectively. When the catalytic efficiencies are compared, soluble isoform of rat intestinal BChE became increasingly efficient as the size of the acyl portion of the substrate increases; BTCh > PTCh > ATCh. Differently, the enzyme showed substrate inhibition with benzoylcholine (BzCh) and a kcat/Km value of 21,190 was found. Triton X-100 inhibited more efficiently the rat intestinal BChE soluble isoform than it did the human serum BChE.

Butyrylcholinesterase (BChE)-cocaine binding and the fundamental pathway for BChE-catalyzed hydrolysis of cocaine have been studied by molecular modeling, molecular dynamics (MD) simulations, and ab initio calculations. Modeling and simulations indicate that the structures of the prereactive BChE/substrate complexes for (-)-cocaine and (+)-cocaine are all similar to that of the corresponding prereactive BChE/butyrylcholine (BCh) complex. The overall binding of BChE with (-)-cocaine and (+)-cocaine is also similar to that proposed with butyrylthiocholine and succinyldithiocholine, i.e., (-)- or (+)-cocaine first slides down the substrate-binding gorge to bind to Trp-82 and stands vertically in the gorge between Asp-70 and Trp-82 (nonprereactive complex) and then rotates to a position in the catalytic site within a favorable distance for nucleophilic attack and hydrolysis by Ser-198 (prereactive complex). In the prereactive complex, cocaine lies horizontally at the bottom of the gorge. The fundamental catalytic hydrolysis pathway, consisting of acylation and deacylation stages similar to those for ester hydrolysis by other serine hydrolases, was proposed on the basis of the simulated prereactive complex and confirmed theoretically by ab initio reaction coordinate calculations. Both the acylation and deacylation follow a double-proton-transfer mechanism. The calculated energetic results show that within the chemical reaction process the highest energy barrier and Gibbs free energy barrier are all associated with the first step of deacylation. The calculated ratio of the rate constant (k(cat)) for the catalytic hydrolysis to that (k(0)) for the spontaneous hydrolysis is approximately 9.0 x 10(7). The estimated k(cat)/k(0) value of approximately 9.0 x 10(7) is in excellent agreement with the experimentally derived k(cat)/k(0) value of approximately 7.2 x 10(7) for (+)-cocaine, whereas it is approximately 2000 times larger than the experimentally derived k(cat)/k(0) value of approximately 4.4 x 10(4) for (-)-cocaine. All of the results suggest that the rate-determining step of the BChE-catalyzed hydrolysis of (+)-cocaine is the first step of deacylation, whereas for (-)-cocaine the change from the nonprereactive complex to the prereactive complex is rate-determining and has a Gibbs free energy barrier higher than that for the first step of deacylation by approximately 4 kcal/mol. A further analysis of the structural changes from the nonprereactive complex to the prereactive complex reveals specific amino acid residues hindering the structural changes, providing initial clues for the rational design of BChE mutants with improved catalytic activity for (-)-cocaine.

Human serum butyrylcholinesterase (BChE) has been converted into a stable but less active desensitized form when heated at 45°C for 24 h. The desensitized BChE follows Michaelis-Menten kinetics, whereas native enzyme exhibits slightly negative cooperativity with respect to butyrylthiocholine binding. In this study, we investigated the effects of Ni2+, Co2+, and Mn2+ on the desensitized BChE. It is found that all three ions were noncompetitive inhibitors of the desensitized BChE, and K i values have been determined as 7.816±1.060 mM, 48.722±4.635 mM, and 84.795±5.249 mM for Ni2+, Co2+, and Mn2+, respectively. In our previous study, these ions were linear mixed-type inhibitors of the native BChE. This finding confirms that desensitized BChE changes to a different conformation than native BChE. From the comparison of K i values of the trace elements, it can be said that Ni2+ is a more effective inhibitor of the desensitized BChE than Co2+ and Mn2+.

In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BuChE), we employed site-directed mutagenesis to modify the catalytic triad residue Ser198, the acyl pocket Leu286 and adjacent Phe329 residues, and Met437 and Tyr440 located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and Km values towards butyrylthiocholine and IC50 values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso-OMPA) were determined. Substitution of Ser198 by cysteine and Met437 by aspartate nearly abolished activity, and other mutations of Ser198 completely abolished it. Tyr440 and Leu286 mutants remained active, but with higher Km and IC50 values. Rates of inhibition by DFP were roughly parallel to IC50 values for several Leu286 mutants. Both Km and IC50 values increased for Leu286 mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe329 displayed unmodified Km values toward butyrylthiocholine, but up to 10-fold decreased IC50 values for DFP, iso-OMPA, and echothiophate. These findings add Tyr440 and Phe329 to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BuChE, and foreshadow the design of recombinant BuChEs with tailored scavenging properties.

Butyrylcholinesterase (BChE) is inhibited by the plant growth regulator (2-chloroethyl)phosphonic acid (ethephon) as observed 25 years ago both in vitro and in vivo in rats and mice and more recently in subchronic studies at low doses with human subjects. The proposed mechanism is phosphorylation of the BChE active site at S198 by ethephon dianion. The present study tests this hypothesis directly using [(33)P]ethephon and recombinant BChE (rBChE) with single amino acid substitutions and further evaluates if BChE is the most sensitive esterase target in vitro and with mice in vivo. [(33)P]Ethephon labels purified rBChE but not enzymatically inactive diethylphosphoryl-rBChE (derivatized at S198 by preincubation with chlorpyrifos oxon) or several other esterases and proteins. Amino acid substitutions that greatly reduce rBChE sensitivity to ethephon are G117H and G117K in the oxyanion hole (which may interfere with hydrogen bonding between glycine-N-H and ethephon dianion) and A328F, A328W, and A328Y (perhaps by impeding access to the active site gorge). Other substitutions that do not affect sensitivity are D70N, D70K, D70G, and E197Q which are not directly involved in the catalytic triad. The effect of pH and buffer composition on inhibition supports the hypothesis that ethephon dianion is the actual phosphorylating agent without activation by divalent cations. Human plasma BChE in vitro and mouse plasma BChE in vitro and in vivo are more sensitive to ethephon than any other esterases detected by butyrylthiocholine or 1-naphthyl acetate hydrolysis in native-PAGE. All mouse liver esterases observed are less sensitive than plasma BChE to ethephon in vitro and in vivo. More than a dozen other esterases examined are 10-100-fold less sensitive than BChE to ethephon. Thus, BChE inhibition continues to be the most sensitive marker of ethephon exposure.

Here we report the results of a study conducted to elucidate the enzymological characteristics of lizard cholinesterases (ChEs) in order to use them as potential biomarkers for pesticide exposure. Serum and brain tissue of the lizard Gallotia galloti were used as ChE sources and in vitro assays were performed to identify acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities. The pH, substrate concentration, and specificity for ChE assays as well as the response of serum BChE to the reactivating agent pyridine-2-aldoxime methochloride (2-PAM) were also investigated in order to assess the possibilities of this methodology in biomonitoring programs. By the use of selective substrates and the inhibitor tetraisopropyl pyrophosphoramide, AChE and BChE activities were identified in lizard serum, while brain contained solely AChE. Likewise, butyrylthiocholine iodide was the optimum substrate for determining BChE activity and acetylthiocholine iodide for assaying both serum and brain AChE activities. The optimal ranges of pH and substrate concentrations were 7.5 to 8.0 and 5 to 10 mM, respectively. Serum was incubated with different doses of the organophosphorus (OP) compounds dichlorvos and paraoxon and subsequently incubated in the presence of two concentrations of 2-PAM (2 x 10(-2) or 2 x 10(-4) M). Reactivation rate of phosphorylated BChE was related to the degree of inhibition of BChE and the dose of 2-PAM. It was found that a 90-min incubation time with 2 x 10(-4) M of 2-PAM satisfactorily increased the OP-inhibited BChE activity. The enzymological properties of serum BChE activity and its in vitro reactivation in the presence of 2-PAM represent the initial justification for its use in monitoring OP contamination in the field.

We describe three catalytic cholinesterase-like catalytic antibodies (Ab1), as well as anti-idiotypic (Ab2) and idiotypic (Ab3) antibodies, to one of the Ab1s. The Ab1s were raised against the human erythrocyte acetylcholinesterase (AChE), and are unusual in that they both recognise and resemble acetylcholinesterase in their catalytic activity. No contamination of the antibody preparations with either acetylcholinesterase or butyrylcholinesterase (BChE) was found. None of the Ab2s showed catalytic activity, whereas four Ab3s did (an incidence of 1.26% of all Ab3s). Although there is considerable resemblance between Ab1s and Ab3s, there are significant differences between the two groups. All the antibodies were inhibited by phenylmethylsulphonyl fluoride (PMSF), indicating the presence of a serine residue in their active sites, and were inhibited by the cholinesterase active site inhibitors iso-OMPA and pyridostigmine, suggesting the similarity of the sites to those of cholinesterases. The Ab3s resemble the Ab1s in their ability to hydrolyse both acetyl and butyrylthiocholine (BTCh). However, the Ab3s appear to be better catalysts, having significantly reduced K(m) values (for acetyl, but not for butyrylthiocholine) and increased turnover numbers (K(cat)), rate enhancements (K(cat)/K(uncat)) and K(cat)/K(m) ratios, for both substrates, although these values by no means approach those of the natural enzymes. The Ab1s appear to have structures resembling the anionic sites of cholinesterases, as shown by their reaction with the anionic site inhibitors (edrophonium and tetramethylammonium). No such reactions were observed in the Ab3s. None of the antibodies show evidence of the sites resembling the peripheral anionic site (PAS) of acetylcholinesterase. All the antibodies recognise, to varying degrees, the peripheral anionic site of acetylcholinesterase. This was shown by their ability to inhibit acetylcholinesterase, to compete with peripheral site inhibitors, and to block acetylcholinesterase-mediated cell adhesion, a property of this site. The results indicate idiotypic mimicry of a catalytic antibody's active site, and suggest that the development of the catalytic activity in the anti-acetylcholinesterase antibodies may be related to the structural features of the peripheral anionic site of acetylcholinesterase.

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was solubilized from cultured Caco-2 cells. It was established that this enzyme activity is acetylcholinesterase by substrate specificity (acetylthiocholine, acetyl-β-methylthiocholine>propionylthiocholine>butyrylthiocholine), substrate inhibition, and specificity of inhibitors (BW284c51>iso-OMPA). The acetylcholinesterase activity increased proportional to the degree of differentiation of the cells. Most of the enzyme was membrane bound, requiring detergent for solubilization, and the active site faced the external fluid. Only one peak of activity, which corresponded to a monomeric form, could be detected on linear sucrose density gradients. The sedimentation of this form of the enzyme was shifted depending on whether Triton X-100 or Brij 96 detergent was used. These results indicate that the epithelial-derived Caco-2 cells produce predominantly an amphiphilic, monomeric form of acetylcholinesterase that is bound to the plasma membrane and whose catalytic center faces the extracellular fluid.

Hydrolysis of the neutral substrate N-methylindoxyl acetate (NMIA) by wild-type human butyrylcholinesterase (BuChE) and peripheral site mutants (D70G, Y332A, D70G/Y332A) was found to follow the Michaelis-Menten kinetics. K(m) was 0.14 mM for wild-type, and 0.07-0.16 mM for D70G, Y332A and D70G/Y332A, indicating that the peripheral site is not involved in NMIA binding. The values of k(cat) were of the same order for all enzymes: 12,000-18,000 min(-1). Volume changes upon substrate binding (-DeltaV(K(m))) and the activation volumes (DeltaV++(k(cat)) associated with hydrolysis of NMIA were calculated from the pressure dependence of the catalytic constants. Values of -DeltaV(K(m)) indicate that NMIA binds to an aromatic residue, presumed to be W82, the active site binding locus. Binding is accompanied by a release of water molecules from the gorge. Residue 70 controls the number of water molecules that are released upon substrate binding. The values of DeltaV++(k(cat)), which are positive for wild-type and faintly positive for D70G, clearly indicate that the catalytic steps are accompanied by re-entry of water into the gorge. Results support the premise that residue D70 is involved in the conformational stabilization of the active site gorge and in control of its hydration. A slow transient, preceding the steady state, was seen on a time scale of several minutes. The induction time rapidly increased with NMIA concentration to reach a limit at substrate saturation. Much shorter induction times (1 min) were seen for hydrolysis of benzoylcholine (BzCh) by wild-type BuChE and for hydrolysis of butyrylthiocholine (BuSCh) by the active site mutants E197Q and E197Q/G117H. This slow transient was interpreted in terms of hysteresis without kinetic cooperativity. The hysteretic behavior of BuChE results from a slow conformational equilibrium between two enzyme states E and E'. NMIA binds only to the primed form E'. Kosmotropic salts and hydrostatic pressure were found to shift the equilibrium toward E'. The E--E' transition is accompanied by a negative activation volume (DeltaV++(0)= -45+/-10 ml/mol), and the E' form is more compact than E. Hydration water in the gorge of E' appears to be more structured than in the unprimed form.

The rat is the model animal for toxicity studies. Butyrylcholinesterase (BChE), being sensitive to inhibition by some organophosphorus and carbamate pesticides, is a biomarker of toxic exposure. The goal of this work was to characterize the purified rat BChE enzyme. The cDNA sequence showed eight amino acid differences between the active site gorge of rat and human BChE, six clustered around the acyl binding pocket and two below the active site serine. A prominent difference in rat was the substitution of arginine for leucine at position 286 in the acyl pocket. Wild-type rat BChE, the mutant R286L, wild-type human BChE, and the mutant L286R were expressed in CHO cells and purified. Arg286 was found responsible for the resistance of rat BChE to inhibition by Triton X-100. Replacement of Arg286 with leucine caused the affinity for Triton X-100 to increase 20-fold, making it as sensitive as human BChE to inhibition by Triton X-100. Wild-type rat BChE had an 8- to 9-fold higher Km for the positively charged substrates butyrylthiocholine, acetylthiocholine, propionylthiocholine, benzoylcholine, and cocaine compared with wild-type human BChE. Wild-type rat BChE catalyzed turnover 2- to 7-fold more rapidly than human BChE, showing the highest turnover with propionylthiocholine (201,000 min−1). Human BChE does not reactivate spontaneously after inhibition by echothiophate, but rat BChE reactivates with a half-life of 4.3 hr. Human serum contains 5 mg/L of BChE and 0.01 mg/L of AChE. Male rat serum contains 0.2 mg/L of BChE and approximately 0.2 mg/L of AChE.

Oligohaline copepods such as Eurytemora affinis are widespread in estuaries of northwestern Europe. These minute crustaceans are highly sensitive to contamination and thus serve as useful bioindicators for the monitoring of pollutant effects. The use of decreased cholinesterase (ChE) activity as a sublethal biomarker of exposure to neurotoxic compounds supposes that ChE has been defined in copepods. This study reports the partial purification and characterization of ChE extracted from E . affinis. Analysis by non-denaturing PAGE and by isoelectric focusing indicated that the enzyme is probably a single dimeric form of 140 KDa, with a p I of 6.2. This enzyme is likely an acetylcholinesterase (AChE) since it hydrolyzes acetylthiocholine iodide at a higher rate than other substrates, such as butyrylthiocholine and propionylthiocholine, at pH 7.0 and 25 °C, and is inhibited by eserine but not by iso-OMPA. The enzyme exhibited high sensitivity to some of the various pollutants tested. The kinetic properties of this ChE were compared with those of other invertebrate ChEs.

The influence of several variables such as sample and reagent storage, anticoagulants, reaction temperature, pH, and substrate concentration on whole blood cholinesterase determination was studied. Storage of nondiluted whole blood samples at room temperature or under refrigeration (4 C) was adequate for short-term storage (3 days to 2 weeks). However, freezing would be more appropriate for long-term storage (≥1 month), and successive thawing and freezing did not produce any loss of cholinesterase activity. All reagents (2,2′-dithiodipyridine as chromophore and acetylthiocholine and butyrylthiocholine as substrates) were stable for 3 months when frozen. Heparin and ethylenediaminetetraacetic acid were the most suitable anticoagulants for whole blood acetylcholinesterase and butyrylcholinesterase determination, because citrate yielded lower acetylcholinesterase values and fluoride inhibited butyrylcholinesterase. Increases in reaction temperature and pH yielded higher cholinesterase values but also increased nonenzymatic substrate hydrolysis. Higher cholinesterase and nonenzymatic substrate hydrolysis values were obtained as higher substrate concentrations were used.

Butyrylcholinesterase purified from human serum as 6600-fold was heated at 37 degrees, 40 degrees, 45 degrees, and 50 degrees C for 24 hr. It was observed that the enzyme heated at 45 degrees C for 24 hr converted to a stabilized form and followed Michaelis-Menten kinetics, whereas the enzyme samples, heated at the other temperatures for 24 hr, shown negative cooperativity with respect to its substrate, butyrylthiocholine. Even the sample heated at 45 degrees C for 12 hr shown negative cooperativity. On the contrary to the heated enzyme at 40 degrees C for 24 hr, the heated enzyme at 45 degrees C for 24 hr could not be reactivated when it was kept at 4 degrees C for 24 hr. In the kinetic studies, it was found that substrate analogs choline and benzoylcholine inhibited both the native enzyme and the enzyme heated at 45 degrees C for 24 hr competitively, whereas succinylcholine was the partial competitive inhibitor of native enzyme but the pure competitive inhibitor of the heated enzyme.

Tetraalkylammonium (TAA) salts are well known reversible inhibitors of cholinesterases. However, at concentrations around 10 mm, they have been found to activate the hydrolysis of positively charged substrates, catalyzed by wild-type human butyrylcholinesterase (EC 3.1.1.8) [Erdoes, E.G., Foldes, F.F., Zsigmond, E.K., Baart, N. & Zwartz, J.A. (1958) Science128, 92]. The present study was undertaken to determine whether the peripheral anionic site (PAS) of human BuChE (Y332, D70) and/or the catalytic substrate binding site (CS) (W82, A328) are involved in this phenomenon. For this purpose, the kinetics of butyrylthiocholine (BTC) hydrolysis by wild-type human BuChE, by selected mutants and by horse BuChE was carried out at 25 °C and pH 7.0 in the presence of tetraethylammonium (TEA). It appears that human enzymes with more intact structure of the PAS show more prominent activation phenomenon. The following explanation has been put forward: TEA competes with the substrate at the peripheral site thus inhibiting the substrate hydrolysis at the CS. As the inhibition by TEA is less effective than the substrate inhibition itself, it mimics activation. At the concentrations around 40 mm, well within the range of TEA competition at both substrate binding sites, it lowers the activity of all tested enzymes.

The action of a potent tricyclic cholinesterase inhibitor ethopropazine on the hydrolysis of acetylthiocholine and butyrylthiocholine by purified horse serum butyrylcholinesterase (EC 3.1.1.8) was investigated at 25 and 37 degrees C. The enzyme activities were measured on a stopped-flow apparatus and the analysis of experimental data was done by applying a six-parameter model for substrate hydrolysis. The model, which was introduced to explain the kinetics of Drosophila melanogaster acetylcholinesterase [Stojan et al. (1998) FEBS Lett. 440, 85-88], is defined with two dissociation constants and four rate constants and can describe both cooperative phenomena, apparent activation at low substrate concentrations and substrate inhibition by excess of substrate. For the analysis of the data in the presence of ethopropazine at two temperatures, we have enlarged the reaction scheme to allow primarily its competition with the substrate at the peripheral site, but the competition at the acylation site was not excluded. The proposed reaction scheme revealed, upon analysis, competitive effects of ethopropazine at both sites; at 25 degrees C, three enzyme-inhibitor dissociation constants could be evaluated; at 37 degrees C, only two constants could be evaluated. Although the model considers both cooperative phenomena, it appears that decreased enzyme sensitivity at higher temperature, predominantly for the ligands at the peripheral binding site, makes the determination of some expected enzyme substrate and/or inhibitor complexes technically impossible. The same reason might also account for one of the paradoxes in cholinesterases: activities at 25 degrees C at low substrate concentrations are higher than at 37 degrees C. Positioning of ethopropazine in the active-site gorge by molecular dynamics simulations shows that A328, W82, D70, and Y332 amino acid residues stabilize binding of the inhibitor.

The benthic mollusk Scapharca inaequivalvis was collected in spring 1999 from three areas of the northern Adriatic Sea. From the mollusk, molecular forms of acetylcholinesterase (AChE), consisting of two prevailing spontaneously soluble (SS) forms present in the blood, were obtained. These forms are a globular tetramer (SSG4), a dimer (SSG2) of catalytic subunits, and a minor amphiphilic globular dimer (low-salt Triton [LST] G2) phosphatidylinositol tailed. All SS and LST forms, partially purified by affinity chromatography, are AChEs with a marked substrate specificity for acetylthiocholine and poor hydrolysis with butyrylthiocholine. They are poorly inhibited by carbamate eserine and show a different pattern of inhibition by organophosphate diisopropylfluorophosphate (DFP), with totally resistant SS forms from clams collected from the two stations nearest the Po River. Acetylcholinesterase SS and LST forms are expressed at highest, lowest, and middle levels in clams collected from the northern station, closer to the Po delta, and from the two more southern ones, respectively. The possibility that the expression pattern of AChE forms is due to effects of single or mixed classes of chemical pollutants is discussed.

A Cholinesterase was isolated and purified (65-fold) from roots of Bengal gram (Cicer arietinum L.) seedlings. It hydrolyzes acetylcholine and acetylthiocholine more readily than propionylthiocholine or butyrylthiocholine. The enzyme has high affinity for acetylthiocholine (Km=150 μm) and is inhibited by animal anticholinesterases, neostigmine and physostigmine, and by Phosfon-D, a plant growth retardant.