Effet de solvants sur l'acetylcholinestérase

Ronzani, N.

doi:10.1016/s0040-4039(00)79249-5

Cited by 9 publications

(5 citation statements)

References 11 publications

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“…The inhibition of AChE by tacrine, gallamine and compound 1 was investigated in the presence of 6% v ⁄ v MeCN (corresponding to a concentration of 1.15 m), and the results are shown in Figs 2B,3B,4 and Table 1. Even without the addition of inhibitors, our research showed that the presence of MeCN reduced the rate of enzyme-catalysed substrate conversion, which is in accordance with several literature reports on soluble and immobilized AChE from E. electricus [75][76][77][78][79][80]. At the highest substrate concentration used in our experiments, [S] = 2250 lm, the absolute enzyme activity without MeCN was 0.251 ± 0.052 min )1 (n = 8), whereas the addition of 6% MeCN resulted in a decrease in the rate to 0.089 ± 0.020 min )1 (n = 8), i.e.…”

Section: Influence Of Mecn On the Inhibition Of Achesupporting

confidence: 92%

“…The latter K m value was calculated on the basis of a competitive mode of inhibition suggested for MeCN and K I = 0.16 m [75,76]. For competitive inhibitors, K I can be calculated according to the equation K I = [I]/[( K m ′/ K m )−1], where K m ′ is the Michaelis constant in the presence of a certain amount of inhibitor [75,76]. Applying this equation to our experiments and using mean values of the data shown in Table 1 ( K m ′ = 666 μ m at [MeCN] = 1.15 m , K m = 118 μ m without MeCN), we calculated an equivalent K I value of 0.25 m for MeCN.…”

Section: Resultsmentioning

confidence: 99%

“…A similar result was found in a study by Ronzani [76], where K m values of 85 and 750 μ m were determined for the AChE‐catalysed conversion of ATCh in the absence and presence of 6.5% MeCN, respectively. The latter K m value was calculated on the basis of a competitive mode of inhibition suggested for MeCN and K I = 0.16 m [75,76]. For competitive inhibitors, K I can be calculated according to the equation K I = [I]/[( K m ′/ K m )−1], where K m ′ is the Michaelis constant in the presence of a certain amount of inhibitor [75,76].…”

Section: Resultsmentioning

confidence: 99%

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Kinetics of inhibition of acetylcholinesterase in the presence of acetonitrile

et al. 2009

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Acetylcholinesterase (AChE, EC 3.1.1.7) is a serine hydrolase [1], which belongs to the a ⁄ b hydrolase family [2,3]. The enzyme hydrolyses a broad range of ester and amide substrates, showing the highest specificity for acetylselenocholine, acetylthiocholine (ATCh) and acetylcholine (ACh) [4]. Substrate cleavage proceeds via a two-step mechanism: acylation of the enzyme, followed by deacylation involving a water molecule [5][6][7]. This process is mediated by the catalytic triad Ser200-His440-Glu327 (Torpedo californica AChE, TcAChE, numbering [8]) located within the active site at the bottom of a 20 Å deep gorge. Substrate binding is facilitated by another component of the active site, the anionic site, which is characterized by several conserved aromatic residues, such as Trp84 and Phe330. These residues have been shown to interact with the quaternary ammonium groups of ACh or ATCh via cation-p interactions [7][8][9][10][11][12]. Further stabilization of the quaternary moiety arises from an electrostatic interaction with the acidic side-chain of Glu199 [7,12]. A second substrate-binding site, the peripheral anionic site (PAS), lies essentially on the The hydrolysis of acetylthiocholine by acetylcholinesterase from Electrophorus electricus was investigated in the presence of the inhibitors tacrine, gallamine and compound 1. The interaction of the enzyme with the substrate and the inhibitors was characterized by the parameters K I , a¢, b or b, K m and V max , which were determined directly and simultaneously from nonlinear Michaelis-Menten plots. Tacrine was shown to act as a mixedtype inhibitor with a strong noncompetitive component (a¢ % 1) and to completely block deacylation of the acyl-enzyme. In contrast, acetylcholinesterase inhibition by gallamine followed the 'steric blockade hypothesis', i.e. only substrate association to as well as substrate ⁄ product dissociation from the active site were reduced in the presence of the inhibitor. The relative efficiency of the acetylcholinesterase-gallamine complex for the catalysis of substrate conversion was determined to be 1.7-25% of that of the free enzyme. Substrate hydrolysis and the inhibition of acetylcholinesterase were also investigated in the presence of 6% acetonitrile, and a competitive pseudo-inhibition was observed for acetonitrile (K I = 0.25 m). The interaction of acetylcholinesterase with acetonitrile and tacrine or gallamine resulted in a seven-to 10-fold increase in the K I values, whereas the principal mode of inhibition was not affected by the organic solvent. The determination of the inhibitory parameters of compound 1 in the presence of acetonitrile revealed that the substance acts as a hyperbolic mixed-type inhibitor of acetylcholinesterase. The complex formed by the enzyme and the inhibitor still catalysed product formation with 8.7-9.6% relative efficiency.

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Section: Influence Of Mecn On the Inhibition Of Achesupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Kinetics of inhibition of acetylcholinesterase in the presence of acetonitrile

et al. 2009

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“…Therefore, the use of enzymes capable of also functioning in the presence of organic solvents would open up a far wider range of applications. ChE can usually function in a manner similar to aqueous solution in low concentrations of organic solvent in a mixed aqueous−organic phase. , To circumvent the problem of hydrophilic solvents stripping the enzymes of essential water, it was suggested that 1−10% water was necessary for enzymatic activity and should be added to the organic solvent for sufficient hydration of the active site of the enzyme . However, a “bienzymatic inhibition organic-phase enzyme electrode” has been developed that can be used to check either OP pesticides or carbamates.…”

Section: Important Role Played By Organic Solventsmentioning

confidence: 99%

“…The development of organic-phase enzyme assays has attracted considerable interest. , It made it possible to extend this analytical field to hydrophobic substrates and inhibitors with decreased interference arising from hydrophilic species. Furthermore, it improves the working life of ChE due to the lower levels of microbial contamination and the higher thermal stability of ChE in organic solvents because chemical and structural changes of enzymes require the existence of water.…”

Section: Important Role Played By Organic Solventsmentioning

confidence: 99%

History and New Developments of Assays for Cholinesterase Activity and Inhibition

2010

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Acetylcholinesterase-based biosensors for quantification of carbofuran, carbaryl, methylparaoxon, and dichlorvos in 5% acetonitrile

et al. 2008

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Amperometric acetylcholinesterase biosensors have been developed for quantification of the pesticides carbofuran, carbaryl, methylparaoxon, and dichlorvos in phosphate buffer containing 5% acetonitrile. Three different biosensors were built using three different acetylcholinesterase (AChE) enzymes-AChE from electric eel, and genetically engineered (B394) and wild-type (B1) AChE from Drosophila melanogaster. Enzymes were immobilized on cobalt(II) phthalocyanine-modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). Each biosensor was tested against the four pesticides. Good operational stability, immobilisation reproducibility, and storage stability were obtained for each biosensor. The best detection limits were obtained with the B394 enzyme for dichlorvos and methylparaoxon (9.6 x 10(-11) and 2.7 x 10(-9) mol L(-1), respectively), the B1 enzyme for carbofuran (4.5 x 10(-9) mol L(-1)), and both the B1 enzyme and the AChE from electric eel for carbaryl (1.6 x 10(-7) mol L(-1)). Finally, the biosensors were used for the direct detection of the pesticides in spiked apple samples.

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Effet de solvants sur l'acetylcholinestérase

Cited by 9 publications

References 11 publications

Kinetics of inhibition of acetylcholinesterase in the presence of acetonitrile

Kinetics of inhibition of acetylcholinesterase in the presence of acetonitrile

History and New Developments of Assays for Cholinesterase Activity and Inhibition

Acetylcholinesterase-based biosensors for quantification of carbofuran, carbaryl, methylparaoxon, and dichlorvos in 5% acetonitrile

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