Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.1.7 (acetylcholinesterase)
28,390 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5'-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coefficient, 13.6 x 10(3)M(-1)cm(-1), as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for determination of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temperature increases, while absorbance maxima decrease. Our recommended molar absorption coefficients at 412 nm are 14.15 x 10(3)M(-1)cm(-1) at 25 degrees C and 13.8 x 10(3)M(-1)cm(-1) at 37 degrees C (0.1M phosphate buffer, pH 7.4). Molar absorption coefficients for other temperatures and wavelengths are included in the paper.
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PMID:Molar absorption coefficients for the reduced Ellman reagent: reassessment. 1253 Dec 9

No comparative information is available concerning the ability of various cholinesterase (ChE) methods to identify succinyldicholine-sensitive patients, purely on the basis of the enzyme activity recorded in serum. Here, we evaluated six different methods for the measurement of ChE activity; 131 subjects were subdivided according to ChE phenotype and, therefore, to succinyldicholine sensitivity. ChE phenotype was determined by measuring dibucaine and fluoride numbers. DNA analysis was also performed to confirm correlation between the phenotype classification used in the study and the ChE genotype. The tested methods were significantly different in their ability to discriminate between the subjects with and without succinyldicholine-sensitive phenotypes. The succinyldithiocholine/5,5'-dithio-bis(2-nitrobenzoate) (DTNB) method showed the highest accuracy (area under the receiver operating characteristic (ROC) curve 0.97) followed by the propionylthiocholine/DTNB method (area under the ROC curve 0.94). On the other hand, the two methods using butyrylthiocholine as substrate and that employing benzoylcholine showed limited clinical utility in discriminating subjects at risk of prolonged apnea (area under the ROC curve < or = 0.9). Using the succinyldithiocholine method, a value < or = 23 U/l was approximately five times as likely to occur in a sensitive individual as in a normal one.
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PMID:Assay using succinyldithiocholine as substrate: the method of choice for the measurement of cholinesterase catalytic activity in serum to diagnose succinyldicholine sensitivity. 1270 41

We have used site-directed mutagenesis and molecular modeling to investigate the inactivation of an invertebrate acetylcholinesterase (AChE), ChE2 from amphioxus, by the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM), creating various mutants, including C310A and C466A, and the double mutants C310A/C466A and C310A/F312I, to assess the relative roles of the two cysteines and a proposal that the increased rate of inactivation in the F312I mutant is due to increased access to Cys310. Our results suggest that both cysteines may be involved in inactivation by sulfhydryl reagents, but that the cysteine in the vicinity of the acyl pocket is more accessible. We speculate that the inactivation of aphid AChEs by sulfhydryl reagents is due to the presence of a cysteine homologous to Cys310. We also investigated the effects of various reversible cholinergic ligands, which bind to different subsites of the active site of the enzyme, on the rate of inactivation by DTNB of wild type ChE2 and ChE2 F312I. For the most part the inhibitors protect the enzymes from inactivation by DTNB. However, a notable exception is the peripheral site ligand propidium, which accelerates inactivation in the wild type ChE2, but retards inactivation in the F312I mutant. We propose that these opposing effects are the result of an altered allosteric signal transduction mechanism in the F312I mutant compared to the wild type ChE2.
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PMID:Inactivation of an invertebrate acetylcholinesterase by sulfhydryl reagents: the roles of two cysteines in the catalytic gorge of the enzyme. 1658 14

Selected mutagenesis of acetylcholinesterase (AChE; EC 3.1.1.7) may enable one to develop more effective scavenging agents in which AChE itself, in combination with an oxime, will complete a catalytic cycle of hydrolysis of the organophosphate by rapid conjugation followed by enhanced nucleophile-mediated hydrolysis of the phosphonyl enzyme conjugate. Through enlargement of the active site gorge of mouse AChE by mutations Y337A, F295L and F297I, we studied continuous enzymatic degradation of S(P)-cycloheptyl methylphosphonyl thiocholine (S(P)-CHMPTCh) in the presence of HI-6. Continuous hydrolysis of S(P)-CHMPTCh was measured spectrophotometrically from thiocholine released during hydrolysis with DTNB as the thiol reagent. The rates of hydrolysis expressed as moles of formed thiocholine per mole of enzyme per minute were 3.3, 0.69, 0.34 and 0.15min(-1) for F295L/Y337A, Y337A, F297I/Y337A and AChE wild-type, respectively. These rates did not depend on the initial S(P)-CHMPTCh concentration range employed. However, by increasing HI-6 concentrations, the rates approached a limiting value, indicating that oxime reactivation is the rate-limiting step in S(P)-CHMPTCh hydrolysis. Our results confirm that a mixture of a mutant enzyme and an oxime might serve as an in vivo catalytic scavenger of organophosphates.
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PMID:Mutation of acetylcholinesterase to enhance oxime-assisted catalytic turnover of methylphosphonates. 1704 38

A sensitive and selective enzymatic kinetic method for the simultaneous determination of mixtures of carbaryl and phoxim pesticides was researched and developed. It was based on the inhibitory effect of the pesticides on acetylcholinesterase (AChE), and the use of 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) as a chromogenic reagent for the thiocholine iodide (TChI) released from the acetylthiocholine iodide (ATChI) substrate. The DTNB-thiocholine reaction was investigated by a spectrophotometric-kinetic approach. The complex rate equation for the formation of the chromogenic product, P, was solved under certain experimental conditions, which enabled the absorbance (A(P), at lambda(max)=412 nm) from the mixtures of the two pesticide inhibitors to be directly related to their concentrations provided the absorbance additivity was followed. The spectra were measured for mixtures of carbaryl and phoxim at different concentrations, and at t=904 s, T=35 degrees C, pH=7.5, c(ATChI)=0.14, and c(AChE)=0.10 mg mL(-1). The detection limits of the enzymatic kinetic spectrophotometric procedures for the determination of the carbaryl and phoxim were 4.7 and 0.59 microg L(-1), respectively. Calibration models for chemometrics methods, such as principal component regression (PCR), partial least squares (PLS) and radial basis function-artificial neural network (RBF-ANN) were constructed and verified with synthetic samples of the mixtures of the two pesticides. The best performing model was based on the RBF-ANN method yielding at approximately 10 ppb analyte concentrations, %RPE(T) (carbaryl=5.2; phoxim=6.5), %Recovery (approx.105%) and %RPE(T) (6.5). Various spiked town-water samples produced recoveries in the range of 98.8-103% for each pesticide.
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PMID:Simultaneous enzymatic kinetic determination of pesticides, carbaryl and phoxim, with the aid of chemometrics. 1738 2

The original Ellman's spectrophotometrical method for cholinesterase activity determination uses 5,5'-dithiobis-2-nitrobenzoic acid (DTNB, Ellman's reagent) as a chromogen and records the level of cholinesterase activity as an increase of absorbance at 412 nm. Although this procedure usually poses no problem, exceptions arise when the concentration of DTNB is far higher than the concentration of acetylthiocholine (ATCH). It was found that the ratio of concentrations of DTNB/ATCH is an important parameter for the ATCH hydrolysis course: high excess of DTNB decreases the hydrolysis rate resulting in a lower measured enzyme activity. Our experiments indicate that this influence of DTNB concentration can be explained by the inhibition of ATCH hydrolysis by DTNB.
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PMID:New findings about Ellman's method to determine cholinesterase activity. 1742 21

Dichlorvos is an acutely toxic organophosphorous pesticide that is known as a classical acetylcholinesterase (AChE; EC 3.1.1.7) inhibitor. Here, the brain AChE from the important Amazonian fish tambaqui (Colossoma macropomum) was assayed in the presence of this insecticide and also of deltamethrin, a classical sodium and potassium channel inhibitor (negative control). Four tissue homogenates were analyzed in triplicate for AChE activity using acetylthiocholine as the substrate and 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) as the color-developing agent. Each tissue homogenate represented pooled brains from five fish. The inhibitory effect of dichlorvos on AChE activities was determined at concentrations from 0.001 to 10 ppm and compared to controls. This effect followed an exponential decay model (y = 9.420 + 26.192e(-x/5.380); r2 = 0.989), presenting IC50 (the concentration of dichlorvos that is required for 50% of AChE inhibition) of 0.081 ppm (0.368 micromol/L). No effect was observed for the deltamethrin, and the concentration 0.0452 micromol/L of dichlorvos was significantly different from this control. These results suggest that tambaqui brain AChE can be proposed as a biomarker for dichlorvos and can be used as a tool for aquatic environment monitoring.
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PMID:Effect of dichlorvos on the acetylcholinesterase from tambaqui (Colossoma macropomum) brain. 1766 85

Previously we used site-directed mutagenesis, in vitro expression, and molecular modeling to investigate the inactivation of an invertebrate acetylcholinesterase, cholinesterase 2 from amphioxus, by the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). We created the mutants C310A, C466A, C310A/C466A and C310A/F312I to assess the roles of the two cysteines and a proposal that the increased rate of inactivation previously found in an F312I mutant was due to increased access of sulfhydryl reagents to Cys310. Our results indicated that both of the cysteines could be involved in inactivation by sulfhydryl reagents, but that the cysteine near the acyl pocket was more accessible. We speculated that the inactivation of aphid AChEs by sulfhydryl reagents was due to the presence of a cysteine homologous to Cys310 and proposed that this residue could be a target for a specific insecticide. Here we reconsider this proposal.
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PMID:Inactivation of an invertebrate acetylcholinesterase by sulfhydryl reagents: a reconsideration of the implications for insecticide design. 1838 63

Red blood cell AChE (RBC-AChE) and plasma BChE can be used as sensitive biomarkers to detect exposure to OP nerve agents, pesticides, and cholinergic drugs. In a comparative study, RBC-AChE and serum BChE activities in whole blood was obtained from forty seven healthy male and female human volunteers, and then exposed separately ex vivo to three OP nerve agents (soman (GD), sarin (GB) and VX) to generate a wide range of inhibition of AChE and BChE activity (up to 90% of control). These samples were measured using four different ChE assays: (i) colorimetric microEllman (using DTNB at 412 nm), (ii) Test-mate ChE field kit (also based on the Ellman assay), (iii) Michel (delta pH), and (iv) the Walter Reed Army Institute of Research Whole Blood (WRAIR WB) cholinesterase assay. The WRAIR assay is a modified Ellman method using DTP at 324 nm (which minimizes hemoglobin interference and improves sensitivity), and determines AChE and BChE in a small whole blood sample simultaneously. Scatter plots of RBC-AChE activities were determined using the WRAIR ChE assay versus the micro-Ellman, Test-mate and Michel after exposure to varying concentrations of soman, sarin and VX. Regression analyses yielded mostly linear relationships with high correlations (r2 = 0.83-0.93) for RBC-AChE values in the WRAIR assay compared to the alternate methods. For the plasma BChE measurements, individual human values were significantly more variable (as expected), resulting in lower correlations using WRAIR ChE versus the alternate assays (r2 values 0.5 - 0.6). To circumvent the limitations of simple correlation analysis, Bland and Altman analysis for comparing two independent measurement techniques was performed. For example, a Bland and Altman plot of the ratio of the WRAIR whole blood AChE and Michel AChE (plotted on the y-axis) vs. the average of the two methods (x-axis) shows that the majority of the individual AChE values are within +/- 1.96 S.D. of the mean difference, indicating that the two methods may be used interchangeably with a high degree of confidence. The WRAIR ChE assay can be thus be used as a reliable inter-conversion assay when comparing results from laboratory-based (Michel) and field-based (Test-mate ChE kit), which use different methodology and report in different units of AChE activity.
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PMID:Advantages of the WRAIR whole blood cholinesterase assay: comparative analysis to the micro-Ellman, Test-mate ChE, and Michel (DeltapH) assays. 1855 83

A sensitive and selective kinetic enzymatic method for the determination of N-methylcarbamate pesticides is presented. It is based on their inhibitory effect on electric eel acetylcholinesterase and the use of 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) as chromogenic reagent for the thiocholine released from the acetylthiocholine iodide substrate. The fast DTNB-thiocholine reaction is monitored photometrically by the stopped-flow technique. Carbaryl, propoxur and carbofuran can be determined at concentrations in the ranges 6.5-120, 2-15 and 0.1-5.0 ng/ml, respectively, by the proposed method. An interference study was also reported.
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PMID:Enzymatic determination of N-methylcarbamate pesticides at the nanomolar level by the stopped-flow technique. 1896 97


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