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The butyrylcholinesterase activity in human blood is an important marker of healthiness. Either level of this enzyme – increased or otherwise – can indicate disease or intoxication. The most popular technique to assess the cholinesterase activity is the detection of thiocholine, a product of enzymatic hydrolysis of butyrylthiocholine. In this work, Ag-paste was used as SERS-substrate and a new technique was created for thiocholine detection. As explained below, the designed technique facilitates also determination of butyrylcholinesterase (BChE) activity both in the buffer and in spike solution with human plasma. Ellman’s assay was used as a reference method, a good correlation between spectrophotometric detection and Raman detection was shown.

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.

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.

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

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.

Sericostoma vittatum is a caddisfly species, endemic to the Iberian Peninsula, proposed as a biomonitor species for lotic ecosystems. Since inhibition of cholinesterases[U+05F3] (ChE) activity has been used to evaluate the exposure of macroinvertebrates to organophosphates and carbamate pesticides, this work intended to characterize the ChE present in this species so their activity can be used as a potential biomarker of exposure. Biochemical and pharmacological properties of ChE were characterized in this caddisfly species using different substrates (acetylthiocholine iodide, propionylthiocholine iodide, and butyrylthiocholine iodide) and selective inhibitors (eserine sulfate, BW284c51, and iso-OMPA). Also, the in vitro effects of two insecticides (carbaryl and chlorantraniliprole) and two psychiatric drugs (fluoxetine and carbamazepine) on ChE activity were investigated. The results suggest that S. vittatum possess mainly AChE able to hydrolyze both substrates acetylthiocholine and propionylthiocholine since: (1) it hydrolyzes the substrate acetylthiocholine and propionylcholine at similar rates and butyrylthiocholine at a much lower rate; (2) it is highly sensitive to eserine sulfate and BW284c51, but not to iso-OMPA; and (3) its activity is inhibited by excess of substrate, a characteristic of typical AChE. in vitro inhibitions were observed only for carbaryl exposure while exposure to chlorantraniliprole and to relevant environmental concentrations of psychiatric drugs did not cause any significant effect on AChE activity. This study suggests that AChE activity in caddisflies can indeed be used to discriminate the effects of specific insecticides in monitoring programs. The use of non-target species such as caddisflies in ecotoxicological research in lotic ecosystems is also discussed.

Several studies indicate that more than one cholinesterase form may be present in the blood of mammals. In this study the predominant plasma cholinesterase activity, the physiological cholinesterase activity as well as cholinesterase sex-dependent changes in non-exposed individuals of rabbit have been established. Plasma cholinesterase was characterized using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three cholinesterase inhibitors (eserine sulfate, BW284C51 and iso-OMPA). The results indicated that propionylthiocholine was the preferred substrate by plasma cholinesterase followed by acetylthiocholine and butyrylthiocholine, and the predominant enzymatic activity was acetylcholinesterase. Physiological plasma cholinesterase activity was 198.9±5.8 nmol/min/ml for male and 205.2±5.0 nmol/min/ml for female using acetylthiocholine as substrate. Thus, sex had no significant effect on the physiological cholinesterase activity (p>0.05). In addition, the in vivo and in vitro sensitivity of plasma cholinesterase to diazinon was also investigated. In rabbits exposed to single doses of diazinon (25 or 125 mg/kg) the higher inhibitions of plasma cholinesterase were reached 9 h after oral administration (53% and 87% inhibition, respectively). Cholinesterase activity significantly recovered up to values similar to pre-administration between 3 and 7 d depending on the administered dose and sex of the animals. Plasma cholinesterase activity decreased to 24%, 53% and 74% of the initial activity at 9 h of in vitro exposure to 1.25, 3.13 and 6.25 mg/l of diazinon, respectively, and it remained steadily depressed throughout the experimental period (10 d). This study has demonstrated the sensitivity of cholinesterase activity in plasma of rabbits following both in vivo and in vitro exposure to sub-lethal concentrations of diazinon.

Competing substrate kinetic analysis of human butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) from the time-course of enzyme-catalyzed substrate hydrolysis, using spectrophotometric assays is described. This study is based on the use of a chromogenic reporter “visible” substrate (substrate A), whose complete hydrolysis time course is retarded by a competing “invisible” substrate (substrate B). For BChE, four visible substrates were used, two thiocholine esters, benzoylthiocholine and butyrylthiocholine, and two aryl-acylamides, o-nitro trifluoro acetaminide and 3-(acetamido)-N,N,N-trimethylanilinium. Three different competing invisible substrates were used, phenyl acetate, acetylcholine and butyrylcholine. For AChE, two visible substrates were used, acetylthiocholine and 3-(acetamido)-N,N,N-trimethylanilinium. For AChE, acetylcholine was competing with visible substrates. The ratio (R) of bimolecular rate constants, kcat/Km, for all couples of substrates, invisible/visible (B/A) covered all possible limit situations, R ≪ 1, R ≈ 1 and R ≫ 1. The kinetic approach, based on the method developed by Golicnik and Masson allowed determination of binding and catalytic parameters of cholinesterases for both visible and invisible substrates. This analysis was applied to michaelian and non-michaelian catalytic behaviors (activation and inhibition by excess substrate). Reevaluation of catalytic parameters obtained for acetylcholine and butyrylcholine more than 50 years ago was made. The method is fast, reliable, and particularly suitable for poorly soluble substrates and for substrates B when no direct spectrophotometric assays exist. Moreover, replacing substrate B by a reversible inhibitor, mechanism of cholinesterase inhibition was possible to study. It is therefore, useful for screening libraries of new substrates and inhibitors, and/or screening of new cholinesterase mutants. This method can be applied to any other enzymes.

Butyrylcholinesterase (BChE) is an enzyme presented in quite high level in blood plasma where it participates in detoxification reactions. Due to fact that the enzyme is constituted in livers, it is a marker of liver parenchyma function. It can be used for diagnosis of poisoning for e.g., nerve agents or carbofuran and intoxication by some drugs such as rivastigmine. The present experiment is devoted for the creation of new spectrophotometric tests for assay of BChE activity in biological samples. Standard Ellman's method was compared with use of 2,6-dichloroindophenol acetate and indoxylacetate as chromogenic substrates. Maximal velocities and Michaelis constants were calculated for the substrates. Considering calibration, 2,6-dichloroindophenol acetate provided the lowest limit of detection: 1.20×10 −9 kat and a long linear range. All methods were verified using pooled human plasma samples and tested for potential interferents. 2,6-dichloroindophenol acetate is recommended as suitable substrate for BChE assay in clinical diagnostics.

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.

The kinetic behavior of cholinesterases is unconventional. While their activities are higher than expected by classical Michaelis-Menten reaction mechanisms, at intermediate substrate concentrations they show strong inhibition by excess of substrate. To date, the main explanations used for all of their kinetic peculiarities include hindrance of product exit, entropically improved water orientation by a second substrate molecule, and complete blockade of the fully occupied active site. However, with the hydrolysis of butyryl(thio)choline by vertebrate acetylcholinesterase, there are time-dependent and substrate-concentration-dependent decreases in catalytic activity. As the substrate depletion results in the expected downwardly concave shape of the progress curves for product formation at low substrate concentrations, this cannot be the reason for the bending of the linear progress curves at higher substrate concentrations. A good theoretical and practical explanation was reached by including the time-dependent appearance of a non-productive enzyme-substrate complex in the reaction scheme. The slow establishment of this complex appears to be a rare occurrence of incorrect substrate orientation at the bottom of the active site, with this blocked by a second substrate molecule.

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.

Anticholinesterase insecticides constitute a major portion of modern synthetic pesticides and the assessment of cholinesterase (ChE) inhibition is widely used as a specific biomarker for evaluating the exposure of non-target organisms to these pollutants. However, most studies on this biomarker were developed on vertebrates and among invertebrates, gastropod mollusks are rarely used. However, gastropods are important members of aquatic habitats and therefore present a high ecological relevance for freshwater ecosystems. In this context, ChE activities were characterized in two freshwater gastropod mollusks, Potamopyrgus antipodarum and Valvata piscinalis, in order to ascertain their value as sentinel species. Firstly, characterization of ChE activities was performed using different substrates (acetylcholine iodide, butyrylcholine iodide and propionylcholine iodide) and specific inhibitors (eserine, iso-OMPA and BW284c51). Secondly, in vivo effect of a widely used organophosphate insecticide, chlorpyrifos, was tested on ChE activity in both species. Results suggested that P. antipodarum possesses two isoforms of cholinesterases, one isoform which properties are intermediate between an acetyl and a propionyl ChE, and one minor isoform which correspond to a butyryl ChE, while V. piscinalis seems to possess only one isoform which displays typical properties of an acetyl ChE. Chlorpyrifos induced no effect on V. piscinalis ChE. In contrast, P. antipodarum activity was significantly decreased by environmental realistic chlorpyrifos concentrations (2.86 and 14.2 nM) after seven days of contact. The present study suggests that P. antipodarum may be employed as a biological indicator for assessing pesticide contamination.

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.

The best known clinical application of serum BChE assay is to predict abnormally prolonged apnea following the application of the muscle relaxant succinylcholine. The aim of the present study was to assess the frequency of BChE phenotypes and to predict the risk of apnea for those receiving succinylcholine among the residents in western Iran.We examined the frequency of nine BChE phenotypes in 1548 volunteers including 816 males and 732 females with the mean age of 35+/-15 years from an apparently healthy group living in western Iran. The frequencies of BChE phenotypes were determined using BChE activity measurements and by inhibition with dibucaine, fluoride, and the compound Ro2-0683 (Hoffman-La-Roche).The reference range for serum total BChE activity was 4600-14000 U/L (using butyrylthiocholine iodide as substrate). The mean value obtained for men (9030 U/L) was significantly (p0.05) higher than that for women (8550 U/L). The frequencies of four alleles U, A, F, S were calculated to be 0.9826, 0.0165, 0.008 and 0.001, respectively. The frequency of phenotypes of BChE was as follows: normal phenotype (UU) 95.5%, moderate sensitive to succinylcholine including UA,US,UF phenotypes was 3.9% and hypersensitive to succinylcholine (AA, AF, AS, FF, SS) was 0.58%.This study indicates that the population of western Iran has a medium frequency of succinylcholine-sensitive individuals compared to other populations. We suggest that determination of BChE activity and phenotype by the micro automated method is well suited to pre-operative screening and detection of at-risk of prolonged apnea in persons receiving succinylcholine in the healthy population of western Iran.

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.

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.

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.

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 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.

Benactyzine and drofenine are widely used anticholinergic drugs. Benactyzine is used to treat organophosphate poisoning and drofenine acts on smooth muscle to stop muscle spasms. Both of these drugs are esters. After they enter the bloodstream, they will interact with butyrylcholinesterase (BChE; acylcholine acyl hydrolase: EC 3.1.1.8), which has an ability to hydrolyze a wide variety of esters. Therefore, the kinetic analysis of their inhibitory effects on human serum BChE was examined using butyrylthiocholine as substrate. Both drugs were competitive inhibitors of BChE and the Ki values of benactyzine and drofenine were calculated to be 0.010 ± 0.001 and 0.003 ± 0.000 mM, respectively, using the Systat (version 5.03, 1991) nonlinear regression analysis software package. According to these parameters, drofenine is a more potent competitive inhibitor of BChE than benactyzine.

We have previously described a catalytic monoclonal antibody, raised against acetylcholinesterase (AChE) and capable of hydrolysing acetylthiocholine. Here, we describe two more such antibodies. All three antibodies were raised against the same antigen, human erythrocyte AChE, a commercial product purified using the cholinesterase anionic site inhibitor, tetramethylammonium. IgG was purified on Protein A-Sepharose, and lack of contamination with AChE or butyrylcholinesterase (BChE) was demonstrated on sucrose density gradients and immunoassay of the fractions. The antibodies recognised AchE and were capable of hydrolysing acetylthiocholine and the larger butyrylthiocholine substrate, and were inactivated by phenylmethylsulphonyl fluoride (PMSF), indicating a serine residue in the active site. K(m), K(cat), K(cat)/K(uncat) and K(cat)/K(m) values were obtained for both substrates. The active sites of the antibodies were probed with anti-cholinesterases known to react with the active and anionic sites of acetyl- and BChE, and the peripheral anionic site of AChE. The antibodies were inactivated to varying degrees by the BChE inhibitors iso-OMPA, ethopropazine and tetracaine, indicating a less sterically constrained site than AChE and the lack of an acyl-binding pocket. They were also partially inhibited by the AChE-specific inhibitors, BW284c51 and propidium. No peripheral anionic site, as seen in AChE, was observed, shown by the almost complete lack of reaction with fasciculin. All three antibodies appear to have structures resembling the anionic sites of the cholinesterases, seen by their inhibition by quaternary and tricyclic compounds. Further work is required to determine whether the catalytic activity shown by these antibodies is germline-encoded, or is the result of complexation of the antigen with an inhibitor at a peripheral site.

Because of deficiencies in the present treatments for organophosphorus anticholinesterase poisoning, we are attempting to develop a catalytic scavenger that can be administered as prophylactic protection. Currently known enzymes are inadequate for this purpose because they have weak binding and slow turnover, so we are trying to make an appropriate enzyme by protein engineering techniques. One butyrylcholinesterase mutant, G117H, has the desired type of activity but reacts much too slowly. This communication describes an attempt to determine the reason for the slow reaction so that a more efficient enzyme might be designed. The results indicate that the mutation at residue 117 has resulted in a distortion of the transition state of the reaction of organophosphorus compounds with the active site serine. This information will be used to develop other mutants that avoid transition state stabilization sites.

The rate of acetylcholine hydrolysis of mammalian heart muscle influences cardiac responses to vagal innervation. We characterized cholinesterases of human left ventricular heart muscle with respect to both substrate specificity and irreversible inhibition kinetics with the organophosphorus inhibitor N,N'-di-isopropylphosphorodiamidic fluoride (mipafox). Specimens were obtained postmortem from three men and four women (61 +/- 5 years) with no history of cardiovascular disease. Myocardial choline ester hydrolyzing activity was determined with acetylthiocholine (ASCh; 1.25 mM), acetyl-beta-methylthiocholine (AbetaMSCh; 2.0 mM), and butyrylthiocholine (BSCh; 30 mM). After irreversible and covalent inhibition (60 min; 25 degrees C) with a wide range of mipafox concentrations (50 nM-5 mM), residual choline ester hydrolyzing activities were fitted to a sum of up to five exponentials using weighted least-squares non-linear curve fitting. In each ease, quality of curve fitting reached its optimum on the basis of a four component model. Final classification of heart muscle cholinesterases was achieved according to substrate hydrolysis patterns (nmol/min per g wet weight) and to second-order organophosphate inhibition rate constants k2 (1/mol per min); one choline ester hydrolyzing enzyme was identified as acetylcholinesterase (AChE; k2/mipafox = 6.1 (+/- 0.8) x 10(2)), and one as butyrylcholinesterase (BChE; k2/mipafox = 5.3 (+/- 1.1) x 10(3)). An enzyme exhibiting both ChE-like substrate specificity and relative resistance to mipafox inhibition (k2/mipafox = 5.2 (+/- 1.0) x 10(-1)) was classified as atypical cholinesterase.

Recombinant acetylcholinesterase from rat brain and two mutants were studied for their hydrolytic activity toward acetyl- and butyrylthiocholine substrates and for their sensitivity toward organophosphate and carbamate inhibitors. Both mutants, a point mutant where F295 was replaced by leucine, and a second mutant where loop PQES was replaced by SG, were designed for increased size of the acyl binding pocket. Wild type and mutant enzymes were expressed in baculovirus-infected insect cells and biochemically characterized. As expected, wild type rat brain acetylcholinesterase hydrolyzed acetylthiocholine, but not butyrylthiocholine. Sensitivity toward small- and medium-sized organophosphate inhibitors like paraoxon-methyl and paraoxon-ethyl was comparable, but bulky organophosphates like ethoprophos were less efficient inhibitors. This tendency applied to carbamates as well, since small carbamoyl moieties like carbofuran and aldicarb were stronger inhibitors than furathiocarb which features a bulky carbamoyl moiety. In contrast to wild type enzyme, both mutants were capable of hydrolyzing butyrylthiocholine. However, k cat / K m toward acetylthiocholine of the F295L mutant was reduced if compared to the wild type enzyme. All five organophosphate and three carbamate inhibitors inhibited mutant F295L more efficiently than the wild type enzyme.

Butyrylcholinesterase (BChE, EC 3.1.1.8) has been purified about 6600-fold from human serum with a procedure including ammonium sulfate fractionation (55–70%) with acid step at pH 4.5 and procainamide–Sepharose 4B affinity chromatography. The purified enzyme exhibited negative cooperativity with respect to butyrylthiocholine (BTCh) binding at pH 7.5. KS was found to be 0.128±0.012 mM. Inhibition kinetics of the enzyme by Cd2+, Zn2+ and Al3+ were studied in detail. The 1/v vs 1/[BTCh] plots in the absence (control plot) and in the presence of different concentrations of cations intersected above 1/[BTCh]-axis. The data were analyzed by means of a nonlinear curve fitting program. The results demonstrated that all of the three cations are the linear mixed-type inhibitors of BChE. Ca2+ and Mg2+ had no effect on the enzyme activity in the experimental conditions. But when the enzyme was inhibited by 0.5 mM Cd2+ or Zn2+, Ca2+ and Mg2+ partially reactivated the inhibited allosteric form of BChE. Results were compared with data obtained from brain BChE purified from sheep.

Hussein, A. S., Grigg, M. E., and Selkirk, M. E. 1999.Nippostrongylus brasiliensis:Characterisation of a somatic amphiphilic acetylcholinesterase with properties distinct from the secreted enzymes.Experimental Parasitology91, 144–150. We have previously determined thatNippostrongylus brasiliensissecretes three monomeric nonamphiphilic (Gna1) variants of acetylcholinesterase (AChE) with broadly similar properties. In this study we have examined AChE expression in somatic extracts ofN. brasiliensisand report the identification of an additional enzyme which is not secreted. The enzyme was resolved by sucrose density gradient centrifugation with a sedimentation coefficient of 10.2 S which was shifted to 9.4 S in the presence of Triton X-100, identifying the enzyme as a tetrameric amphiphilic (Ga4) form. The amphiphilic properties of this enzyme were confirmed by charge-shift electrophoresis, in which migration was accelerated by interaction with sodium deoxycholate. The enzyme showed low activity with butyrylthiocholine, and a Michaelis constant of 91 ± 13 μM for acetylthiocholine was determined. It was highly sensitive to the AChE- specific inhibitor bis (4-allyldimethylammoniumphenyl)pentan-3-one dibromide, with an IC50of 6.5 ± 0.4 μM, but was also inhibited by the butyrylcholinesterase-specific inhibitor tetramonoisopropylpyrophosphortetramide, albeit with a higher IC50of 46.5 ± 6.1 μM. This enzyme can therefore be distinguished from the secreted AChEs by its amphiphilic properties, sedimentation in sucrose gradients, and sensitivity to cholinesterase inhibitors.

Wild-type human butyrylcholinesterase (BuChE) has a non-Michaelian behaviour showing substrate activation with butyrylthiocholine (BTC) as the substrate. The D70G mutant has a catalytic constant identical to that of the wild-type enzyme, but a 10-fold lower affinity for BTC compared to wild-type enzyme, and it does not exhibit activation by excess BTC under conventional conditions. In the present work it was found that addition of polyols or sugars changed the kinetic behaviour of the D70G mutant with BTC. In the presence of 40% sucrose, the D70G mutant enzyme displayed marked activation by excess substrate. Because D70 is hydrogen bonded to Y332, mutants of Y332 were studied. Mutant Y332F had a behaviour similar to that of wild-type BuChE, whereas mutants Y332A, Y332A/D70G and D70G had negligible substrate activation. The behavior of wild-type, Y332F, Y332A and Y332A/D70G did not change in the presence of high concentrations of sugar. Substrate activation has been explained by binding of a second substrate molecule in the peripheral site at D70. The D70G mutant should be incapable of substrate activation, if D70 were the only residue involved in substrate activation. The ability of the D70G mutant to display substrate activation by medium engineering suggests that other residues are involved in initial substrate binding and activation by excess substrate. Osmolyte-induced change in conformation and/or hydration status of Y332 and other solvent-exposed residues may account for the non-Michaelian behaviour of the D70G mutant.

The effects of 20 kHz ultrasound on catalytic activity and structure of the tetramer of wild-type human butyrylcholinesterase (BChE) from plasma and recombinant D70G mutant enzyme were studied at constant temperature. Effects on catalytic properties of both enzymes were investigated by kinetic analysis under ultrasound irradiation using a neutral substrate (o-nitrophenylbutyrate), a positively charged substrate (butyrylthiocholine), and a negatively charged substrate (aspirin). Effects on structure of highly purified wild-type BChE were followed by gel electrophoresis and activity measurements at Vmax after ultrasound treatment. Unlike hydrostatic pressure, mild ultrasound had moderate effects on catalytic parameters of BChE-catalyzed hydrolysis of substrates. For both wild-type and D70G, Km increased slightly with butyrylthiocholine and o-nitrophenylbutyrate under ultrasound irradiation, suggesting that these effects of ultrasound were not due to the periodic variation of pressure but rather to shear forces that took off substrate from the peripheral site and altered diffusion to the active site. By contrast, affinity of the D70G mutant for aspirin slightly increased with ultrasound power, suggesting that ultrasound-induced microstreaming unmasked peripheral residues involved in recognition and initial binding of the negatively charged substrate. Results support the contention that Km is a composite affinity constant, including dissociation constant of the first encounter enzyme-substrate complex on the peripheral site. Small changes in catalytic activity may have resulted from ultrasound-induced subtle conformational changes altering the active site reactivity. Short ultrasound irradiation induced a faint transient enzyme activation, but prolonged irradiation caused partial dissociation of the tetrameric enzyme and irreversible inactivation. Partial dissociation was related to enzyme microheterogeneity, i.e., nicked (C-terminal segment depleted) tetramers were less stable than native tetramers. The resistance of the native tetramer to ultrasound-induced dissociation was ascribed to the existence of an aromatic amino acid array on the apolar side of the C-terminal helical segment of subunits, the four subunits being held together in a four-helix bundle containing the aromatic zipper motifs. Aromatic/aromatic interactions between the four helical segments are thought to be enhanced by ultrasound-generated pressure.

The activity of immobilised butyrylcholinesterase (BChE) was determined potentiometrically by field-effect-transistor (FET), using butyrylthiocholine iodide (BTCh) as a substrate. The enzyme activity increased in the presence of Ca 2 and Mg 2 . Computation of experimental data showed: (1) the increase of enzyme and substrate affinity in the presence of Mg 2 ; and (2) the increase of the rate of formation of hydrolysis products from the enzyme-substrate complex in the presence of Ca 2 and Mg 2 . Positive cooperativity was observed for Mg 2 , the Hill coefficient increased from 1.23 to 2.37 with the increase of Mg 2 concentration from 0.1 to 10 mM. For Ca 2 the coefficient was about 1.0 independently from the concentration. © 1998 Elsevier Science

1. 1. A differential inhibition assay was developed for the quantitative determination of cholinesterase isoenzymes acetylcholinesterase (ACNE; EC 3.1.1.7), cholinesterase (BChE; EC 3.1.1.8), and atypical cholinesterase in small samples of left ventricular porcine heart muscle. 2. 2. The assay is based on kinetic analysis of irreversible cholinesterase inhibition by the organophosphorus compound N,N′-di-isopropylphosphorodiamidic fluoride (mipafox). With acetylthiocholine (ASCh) as substrate (1.25 mM), hydrolytic activities (A) of cholinesterase isoenzymes were determined after preincubation (60 min, 25°C) of heart muscle samples with either saline (total activity, AT), 7μM mipafox (AM1), or 0.8 mM mipafox (AM2): (BChE)=AT -AM1, (AChE)=AM1-AM2, (Atypical ChE)=AM2. 3. 3. The mipafox differential inhibition assay was used to determine the substrate hydrolysis patterns of myocardial cholinesterases with ASCh, acetyl-β-methylthiocholine (AβMSCh), propionylthiocholine (PSCh), and butyrylthiocholine (BSCh). The substrate specificities of myocardial AChE and BChE resemble those of erythrocyte AChE and serum BChE, respectively. Michaelis constants KM with ASCh were determined to be 0.15 mM for AChE and 1.4 mM for BChE. 4. 4. Atypical cholinesterase, in respect to both substrate specificity and inhibition kinetics, differs from cholinesterase activities of vertebrate tissue and, up to now, could be identified exclusively in heart muscle. The enzyme's Michaelis constant with ASCh was determined to be 4.0 mM. 5. 5. The reversible inhibitory effects of physostigmine (eserine) and quinidine on heart muscle cholinesterases were investigated using the differential inhibition assay. With all three isoenzymes, the inhibition kinetics of both substances were strictly competitive. The physostigmine inhibition of AChE was most pronounced (Ki=0.22 μM). Quinidine most potently inhibited myocardial BChE (Ki=35 μM).

An amperometric biosensor for the determination of organophosphorus and carbamate pesticides is described. The biosensor is based on robust, polishable and easily machinable biocomposites containing graphite powder, a non-conducting epoxy resin, the electronic mediator 7,7,8,8-tetracyanoquinodimethane (TCNQ) and the enzyme acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) immobilized on aminated silica particles. Determinations were done with acetylthiocholine (ATCh) or butyrylthiocholine (BTCh) as substrate. Thiocholine produced by enzymatic hydrolysis is oxidized electrocatalytically. The biosensor operates at a potential of 300 mV vs. Ag/AgCl in a pH buffered solution with 0.1 M phosphate and 0.1 M KCl. The decrease rate of the steady-state current after the addition of pesticides was used for the determination. The materials employed in the construction of the reported biosensors allow for a prolonged use of the device if a slight polish of the surface is performed from time to time.

In vitro studies have implicated butyrylcholinesterase (Bche, B.C.3.1.1.8) as the major enzyme for metabolizing cocaine in humans, but little is known about endogenous BChE activity in monkeys and other animals often used in preclinical studies of cocaine. We compared BChE activity in 18 rhesus and 11 squirrel monkeys, using the colorimetric method of Ellman with butyrylthiocholine as substrate, and in vitro cocaine half-life in pooled plasma samples measuring cocaine concentrations over 60 minutes by GC-MS. Rhesus monkeys had a significantly higher plasma BChE activity than squirrel monkeys (8.2 ± 0.5 U/L vs. 2.8 ± 0.5 U/L), and a three-fold shorter in vitro cocaine half-life (20.1 min vs. 60.2 min). BChE activity in rhesus monkeys was comparable to the activity reported in humans. There was no significant influence of age, weight, or prior cocaine exposure. These results indicate that BChE level can vary between species of non-human primates, a factor that should be taken into account when studying drugs such as cocaine which are metabolized by BChE.

The acetylcholinesterase active site consists of a gorge 20 A deep that is lined with aromatic residues. A serine residue near the base of the gorge defines an acylation site where an acyl enzyme intermediate is formed during the hydrolysis of ester substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin 2, a snake neurotoxin, bind and interfere with catalysis. We report here the association and dissociation rate constants for fasciculin 2 interaction with the human enzyme in the presence of ligands that bind to either the peripheral site or the acylation site. These kinetic data confirmed that propidium is strictly competitive with fasciculin 2 for binding to the peripheral site. In contrast, edrophonium, N-methylacridinium, and butyrylthiocholine bound to the acylation site and formed ternary complexes with the fasciculin 2-bound enzyme in which their affinities were reduced by about an order of magnitude from their affinities in the free enzyme. Steady state analysis of the inhibition of substrate hydrolysis by fasciculin 2 revealed that the ternary complexes had residual activity. For acetylthiocholine and phenyl acetate, saturating amounts of the toxin reduced the first-order rate constant kcat to 0.5-2% and the second-order rate constant kcat/Kapp to 0.2-2% of their values with the uninhibited enzyme. To address whether fasciculin 2 inhibition primarily involved steric blockade of the active site or conformational interaction with the acylation site, deuterium oxide isotope effects on these kinetic parameters were measured. The isotope effect on kcat/Kapp increased for both substrates when fasciculin 2 was bound to the enzyme, indicating that fasciculin 2 acts predominantly by altering the conformation of the active site in the ternary complex so that steps involving proton transfer during enzyme acylation are slowed.

Cholinesterase activity was determined in extracts of muscle tissue from 28 species of fish. Flatfish and sea bass muscle were found to be particularly high in activity. Fish muscle cholinesterase from 11 high-activity species was separated from a small amount (1–10%) of acetylcholinesterase and further characterized kinetically. The K M s ranged from 0.077 to 0.93 mM using acetylthiocholine substrate, and from 0.036 to 0.323 mM using butyrylthiocholine substrate. The acetylthiocholine/butyrylthiocholine V max ratio ranged from 0.5 to 3.3. These cholinesterases were extremely sensitive to organophosphate, hypochlorite-activated organothiophosphate and carbamate pesticides.

Acetylcholinesterase (AChE) activity in homogenates of KCM strain western flower thrips, Frankliniella occidentalis (Pergande), was chiefly membrane bound, sensitive to eserine, and insensitive to diisopropyl phosphorofluoridate (DFP). The rate of substrate hydrolysis decreased in the order acetylthiocholine (ASCh)→ propionylthiocholine→ acetyl-β-methylthiocholine (MeSCh)→ butyrylthiocholine (BuSCh). Inhibition by excess substrate was not observed at concentrations up to 8.0 mM. Inhibition by BW 284C51 indicated the presence of two components with different sensitivity to this compound. Butyrylcholinesterase (BuChE) activity, which was considerably higher than AChE activity, was chiefly soluble and sensitive to eserine and DFP and insensitive to BW 284C51. The sulfhydryl reagents p-hydroxymercuribenzoic acid and 5,5′dithiobis-2-nitrobenzoic acid inhibited AChE and BuChE activities. Several organophosphates and carbamates were potent inhibitors of ASCh and BuSCh hydrolysis with the organophosphates being especially active against BuSCh. Substrate specificity and inhibitor sensitivity of AChE and BuChE from UMC strain thrips were similar but not identical to that of KCM thrips. It was suggested that BuChE in western flower thrips might function as a scavenger enzyme to protect AChE from anticholinesterase insecticides. Similarities between the properties of western flower thrips cholinesterases and those reported previously for aphids were striking.

Acetyl- and butyrylcholinesterases (ACHE, BuChE) from various species differ in their substrate specificities and sensitivities to a wide range of inhibitors, yet display conserved sequence, structure and catalytic properties. To determine features that confer these selective properties, residues 58 through 133 of recombinant human BuChE were replaced with the corresponding sequence from human ACHE. The replaced region (>60% identity) spans the Asp70 residue, important for ligand interactions, and the choline binding site, and introduces differences of charge and hydrophobicity in the outer rim and on the surface of the active site gorge. Expressed in microinjected Xenopus laevis oocytes, the resultant chimera retained the catalytic activity, substrate specificity and the Km value toward butyrylthiocholine characteristic of BuChE. Further, it did not acquire substrate inhibition, which is unique to ACHE, although it lost the property of substrate activation, characteristic of BuChE. Moreover, the chimera resembled BuChE in its sensitivity to succinylcholine and physostigmine, but acquired the AChE-like sensitivity to echothiophate and iso-OMPA, and displayed an intermediate pattern of inhibition, more similar to that of AChE than of BuChE, toward bambuterol, dibucaine and BW284C51. These findings demonstrate that the exchanged residues are involved in inhibitor recognition, but not in substrate distinction and in direct catalysis. Furthermore, substrate interaction with the exchanged domain may mediate structural changes leading to substrate activation in BuChE and inhibition in ACHE. The two AChE-specific aromatic tyrosine residues positioned near Asp70 within this region are hence implicated in the peripheral anionic site of cholinesterases, which is involved in the recognition of various ligands.

Pseudocholinesterase (ChE) (acylcholineacylhydrolase, EC 3.1.1.8) has been partially purified (about 270-fold) from sheep brain. The procedure included ammonium sulfate fractionation (20-80%), DEAE-Trisacryl M chromatography and procainamide-Sepharose 4B affinity chromatography. The molecular weight of purified ChE was found to be 290,000 by gel filtration. Kinetic properties of the enzyme have been studied using the substrate analogues choline, succinylcholine and benzoylcholine. It was shown that the inhibition was partially competitive.