Comparison of Two Storage Methods for the Analysis of Cholinesterase Activities in Food Animals

Askar, Kasim Abass; Kudi, A. C.; Moody, A. John

doi:10.4061/2010/904249

Cited by 8 publications

(8 citation statements)

References 22 publications

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“…Briefly, 0.02 ml of sample and 0.24 ml of assay mixture [9.75 ml of 0.1 M sodium phosphate buffer, pH 8.0, containing 1 mM EDTA, and 0.25 ml of 0.2 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB)] were mixed and allowed to stand for 5 min, and then, 0.04 ml of substrate solution was added. The absorbance increase was monitored for 5 min at 410 nm, at 25°C in a plate reader (OptiMax, Molecular Devices, Sunnyvale, CA) [25,26]. There may be some non-enzymic (endogenous) reaction between the sample and the DTNB which may interfere with the analysis.…”

Section: Enzyme Activity Measurementmentioning

confidence: 99%

Purification of Soluble Acetylcholinesterase from Sheep Liver by Affinity Chromatography

Askar

Kudi

Moody

2011

Appl Biochem Biotechnol

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The purpose of this study was to develop a protocol for the purification of acetylcholinesterase (AChE, acetylcholine acetylhydrolase, E.C.3.1.1.7) enzyme and to extend a purification method for further enzyme characterization. A further aim was to study whether the edrophonium's pharmacologic action is due primarily to the inhibition or inactivation of AChE at sites of cholinergic transmission. The purification of a soluble AChE from sheep liver using affinity chromatography on Concanavalin A-Sepharose 4B and edrophonium-Sepharose 6B is studied. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose at flow rate of 0.5 ml/min. AChE is a pivotal enzyme in the cholinergic nervous system. Its primary function is to catalyze hydrolysis of released acetylcholine (ACh) and thus maintain homeostasis of this neurotransmitter in the central and peripheral nervous systems. Hence, AChE is important in both pharmacological and toxicological mechanisms. It was purified 842-fold with a specific activity of 21 U/mg protein. Sodium dodecyl sulfate (SDS) electrophoresis resulted in a monomeric molecular weight of 67.04 kDa, while on gel chromatography using Sephacryl S-200 under nondenaturing conditions to be 201.5 kDa. Based on the molecular weight obtained by gel filtration, the purified AChE was assumed to be a tetrameric form.

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Section: Enzyme Activity Measurementmentioning

confidence: 99%

Purification of Soluble Acetylcholinesterase from Sheep Liver by Affinity Chromatography

Askar

Kudi

Moody

2011

Appl Biochem Biotechnol

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“…Substrate solutions (final concentration 2 mM for PrTChI and PSA, while 1 mM for AcTChI and BuTChI) were prepared and used on the same day and kept on ice during use. Enzyme activities were calculated using an extinction coefficient of 13.6 mM −1 cm −1 for TNB [ 21 ] and are expressed as units (1 U ≡ 1 μ mol ≡ 1000 nmol of substrate hydrolysed per min) per g wet weight of liver homogenate, while for plasma they are expressed as U/mL [ 2 ]. All measurements in this paper were carried out in duplicate.…”

Section: Methodsmentioning

confidence: 99%

“…Enzymes that preferentially catalyse the hydrolysis of ester bonds are classified as esterase (EC 3.1) and generally classified into two subgroups, carboxylesterase (CbE; EC 3.1.1.1) and cholinesterase (ChE; EC 3.1.1.7), depending on their substrate specificity and behaviour towards some inhibitors [ 1 , 2 ]. There are many esterase decreases among carbamate (CB) and organophosphate (OP) compounds, which are the most important in the agriculture and veterinary medicine to control insect invasion; these compounds exert their toxic effects through inhibiting esterase enzyme activities [ 3 – 5 ].…”

Section: Introductionmentioning

confidence: 99%

A Method for Fast Assessment of OP/CB Exposure in the Japanese Quail (Coturnix coturnix japonica) Using Combined Esterases Enzyme Activity as Biomarkers

Abass

2014

Enzyme Research

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The aims of this study were to investigate the presence of different esterase activities in plasma and liver for Japanese quail and to combine determination of both carboxylesterase and cholinesterase as biochemical biomarker in order to identify the effects of carbamate and organophosphate compounds exposure. Carboxylesterase exhibits larger sensitivity to carbamate and organophosphate compounds than to cholinesterase and is present at higher levels. This permitted nature and distribution of carboxylesterase or cholinesterase to be measured. One predominant toxicological form of enzyme level constant in its patterns of motivation and inhibition with cholinesterase was identified in plasma with an apparent Michaelis constant for butyrylthiocholine iodide of 0.394 mM. Carboxylesterase activity in liver was considered by its preferential hydrolysis of the S-phenyl thioacetate. A concentration dependent decrease of carboxylesterase and cholinesterase has demonstrated during in vitro incubation of malathion, parathion, and trichlorfon in the range 0.125–2 mM, while with methomyl was in the range 0.25–4 mM. When quail (n = 15) was exposed orally for 48 h to concentrations of carbamate or organophosphate compounds of 3–200 mg/kg, the percentage inhibition of cholinesterase was in each case larger than that of carboxylesterase and reached statistical significance (P < 0.05) at lower concentrations.

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“…For the measurement of maximal inhibitory concentrations (IC 20, IC 50, and IC 80 ) blood samples were inhibited for 30 min at room temperature 25°C with appropriate concentration malathion compound, depending on preliminary range finding tests [ 2 , 25 ]. Controls were incubated with phosphate buffer pH 8.0 included.…”

Section: Methodsmentioning

confidence: 99%

Experimental Measurements for the Effect of Dilution Procedure in Blood Esterases as Animals Biomarker for Exposure to OP Compounds

Abass

2014

BioMed Research International

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Organophosphate compounds can bind to carboxylesterase, which may lower the concentration of organophosphate pesticides at the target site enzyme, cholinesterase. It is unclear from the literature whether it is the carboxylesterase affinity for the organophosphate and/or the number of carboxylesterase molecules that is the dominant factor in determining the protective potential of carboxylesterase. The fundamental dilutions and kinetic effects of esterase enzyme are still poorly understood. This study aims to confirm and extend our current knowledge about the effects of dilutions on esterases activities in the blood for birds with respect to protecting the enzyme from organophosphate inhibition. There was significantly higher esterases activities in dilution 1 : 10 in the all blood samples from quail, duck, and chick compared to other dilutions (1 : 5, 1 : 15, 1 : 20, and 1 : 25) in all cases. Furthermore, our results also pointed to the importance of estimating different dilutions effects prior to using in birds as biomarker tools of environmental exposure. Concentration-inhibition curves were determined for the inhibitor in the presence of dilutions 1 : 5, 1 : 10, plus 1 : 15 (to stimulate carboxylesterase). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (malathion) were in agreement with the literature, serving to support the use of this assay. Among the thiol-esters dilution 1 : 5 was observed to have the highest specificity constant (k cat/K m), and the K m and k cat values were 176 μM and 16,765 s−1, respectively, for S-phenyl thioacetate ester, while detected in dilution 1 : 15 was the lowest specificity constant (k cat/K m), and the K m and k cat values were 943 μM and 1154 s−1, respectively, for acetylthiocholine iodide ester.

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Comparison of Two Storage Methods for the Analysis of Cholinesterase Activities in Food Animals

Cited by 8 publications

References 22 publications

Purification of Soluble Acetylcholinesterase from Sheep Liver by Affinity Chromatography

Purification of Soluble Acetylcholinesterase from Sheep Liver by Affinity Chromatography

A Method for Fast Assessment of OP/CB Exposure in the Japanese Quail (Coturnix coturnix japonica) Using Combined Esterases Enzyme Activity as Biomarkers

Experimental Measurements for the Effect of Dilution Procedure in Blood Esterases as Animals Biomarker for Exposure to OP Compounds

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