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Acetylcholinesterase of mangrove oyster Crassostrea rhizophorae: A highly thermostable enzyme with promising features for estuarine biomonitoring
Aquatic Toxicology ( IF 4.5 ) Pub Date : 2018-02-12 , DOI: 10.1016/j.aquatox.2018.02.008
Paula Rayane de Souza , Kelma Sirleide de Souza , Caio Rodrigo Dias de Assis , Marlyete Chagas de Araújo , Kaline Catiely Campos Silva , Juliett de Fátima Xavier da Silva , Andreia Cybelle Marques Ferreira , Valdir Luna da Silva , Mônica Lúcia Adam , Luiz Bezerra de Carvalho , Ranilson de Souza Bezerra

Enzyme biomarkers from several aquatic organisms have been used for assessing the exposure to contaminants at sublethal levels. Amongst them, the cholinesterases are commonly extracted from several organisms to evaluate/measure organophosphate and carbamate neurotoxic effects. Acetylcholinesterase (AChE; EC 3.1.1.7) is an enzyme of the group of serine esterases that acts on the hydrolysis of the neurotransmitter acetylcholine allowing the intermittence of the nerve impulses responsible for the neuronal communication. This enzyme is the main target for the action of some pesticides and the inhibition of its activity in bivalve mollusks may be used as biomarker due to their filter-feeding habit. In this context, the present study aimed to characterize physicochemical and kinetic parameters of the AChE extracted from gills and viscera of the oyster Crassostrea rhizophorae and investigate the in vitro effect of pesticides (dichlorvos, diazinon, chlorpyrifos, methyl-parathion, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron and novaluron) in search for assessing its potential as biomarker. Specific substrates and inhibitors evidenced the predominance of AChE in both tissues. The optimum pH found for gills and viscera AChE were 8.0 and 8.5, respectively. The maximum peak of activity occurred at 70 °C for gill AChE and 75 °C for viscera AChE. The enzymes of both tissues presented remarkable thermostability. The Michaelis-Menten constant for both enzymes were 1.32 ± 0.20 mM for gills and 0.43 ± 0.12 mM for viscera. The Vmax values for gills and viscera were 53.57 ± 1.72 and 27.71 ± 1.15 mU/mg, respectively. The enzymes were able to reduce the activation energy to 9.75 kcal mol−1 (gills) and 11.87 kcal mol−1 (viscera) obtaining rate enhancements of 3.57 × 105 and 1.01 × 104, respectively, in relation to non-catalyzed reactions. Among the pesticides under study, the carbamates carbaryl and carbofuran exerted the strongest inhibitory effects on the enzyme activity achieving important degrees of inhibition at concentrations below national and international current regulations. The first observation of the effects of benzoylurea pesticides (diflubenzuron and novaluron) on AChE from mollusks is reported here. The gills AChE of C. rhizophorae showed potential to be specific biomarker for the carbamate carbaryl while the viscera AChE showed it for carbofuran. According to their features, these enzymes may be proposed as promising tools for estuarine monitoring as well as biocomponent of biosensor devices.



中文翻译:

红树林牡蛎Crassostrea rhizophorae的乙酰胆碱酯酶:一种高度热稳定的酶,具有用于河口生物监测的有前途的功能

来自几种水生生物的酶生物标记已用于评估亚致死水平的污染物暴露。其中,胆碱酯酶通常从几种生物中提取,以评估/测量有机磷酸酯和氨基甲酸酯的神经毒性作用。乙酰胆碱酯酶(AChE; EC 3.1.1.7)是丝氨酸酯酶中的一种酶,可作用于神经递质乙酰胆碱的水解,从而使负责神经元通讯的神经冲动间断。该酶是某些农药作用的主要靶标,由于其滤食性习性,在双壳类软体动物中抑制其活性可用作生物标记。在此背景下,本研究旨在表征从牡蛎g和内脏中提取的AChE的理化和动力学参数牡蛎rhizophorae并调查在体外评估其潜在的生物标志物作为在搜索农药(敌敌畏,二嗪农,毒死蜱,甲基对硫磷,双硫磷,甲萘威,克百威,涕灭威,除虫脲和氟酰脲)的效果。特定的底物和抑制剂证明了AChE在两种组织中均占主导地位。发现g和内脏AChE的最佳pH分别为8.0和8.5。ill AChE的最大活性峰出现在70°C,内脏AChE的最大活性峰出现在75°C。两种组织的酶均表现出显着的热稳定性。两种酶的米氏常数分别为1.的1.32±0.20 mM和内脏的0.43±0.12 mM。最大电压g和内脏的数值分别为53.57±1.72和27.71±1.15 mU / mg。这些酶能够将活化能降低到9.75 kcal mol -1(g )和11.87 kcal mol -1(内脏),与非催化反应相比,速率分别提高了3.57×10 5和1.01×10 4。 。在所研究的农药中,氨基甲酸酯西维因和卡百威对酶的活性具有最强的抑制作用,在低于国家和国际现行法规的浓度下仍可达到重要的抑制程度。本文首次报道了苯甲酰脲农药(双氟苯甲隆和去甲灵)对软体动物AChE的影响。C的A AChE 。根瘤菌显示出可能是氨基甲酸酯西维因的特定生物标志物,而内脏AChE则显示出了呋喃丹的特征。根据它们的特征,这些酶可能被提议作为河口监测以及生物传感器设备生物成分的有前途的工具。

更新日期:2018-02-12
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