Development of a new method to assess organophosphate insecticides exposure

自从几种化学品作为有机磷酸盐和碳酸盐抑制剂和重金属抑制剂，以及乙酰胆碱酯酶的组织水平（ACHE）在各种鱼中已经被用作潜在的生物标记器，用于暴露在水生环境中的这些化学品。在订购使AchE更可靠和更有用的生物标记器时，它似乎是一种新的方法来评估有机磷酶暴露，但由于鱼在AchE组织水平的组织水平上的个人差异，将被开发出来。这也是令人难以置信的，因为乙酰胆碱酯在净化、催化性质和对酶的抑制剂敏感性后被表征为良好，从一般的物种到物种不等。不管怎样，那里已经没有普通的新鲜水鱼的特征化乙酰胆碱酯了。在目前的研究中，我们从Koi Carp身体麝香（Cyprinus Carpio）中净化和表征的乙酰纯化的酶具有14，900 μmol·min^<-1>·mg^<-1>蛋白质和催化亚单位的分子质量的高特异性活性，为76.8 kDa。催化特性中的纯化酶的特征、子集特性和抑制剂特性的说明，即Koi Carp麝香中的纯化酶是一种AchE。在第二阶段，我们尝试将特定的多克隆抗体生产到催化子单位的纯化Koi Carp AchE中的催化子单位，是用来开发的新方法来评估有机磷酸盐的暴露的。不幸的是，那个人根本就没有被抓住。

Since several chemicals such as organophosphate and carbamate insecticides and heavy metals inhibit cholinesterases, the tissue levels of acetylcholinesterase(AChE) in various fishes have been used as a potential biomarker for exposure to these chemicals in aquatic environments. In order to make AChE a more reliable and useful biomarker, it seems essential that a new method to assess organophosphate insecticides exposure, which is not affected by individual difference of fish in tissue levels of AChE, be developed. It is also desirable that the AChE is characterized well after purification, since catalytic properties and inhibitor sensitivities of the enzyme to various chemicals vary from species to species in general. However, there has been no characterized AChE of common fresh-water fishes. Thus in the present study, we purified and characterized AChE from body muscle of Koi carp(Cyprinus carpio), one of the fishes used for environmental monitoring in aquatic environment, and tried to develop the new method described above.At the first step, we purified the collagen-tailed asymmetric forms of AChE, the main forms in Koi carp body muscle, 79700-fold. The purified enzyme had a high specific activity of 14900μmol・min^<-1>・mg^<-1> protein and the molecular mass of the catalytic subunit was 76.8kDa. The characterizations of the purified enzyme in catalytic property, substrate specificity and inhibitor specificity demonstrate that the purified AChE from Koi carp muscle is certainly an AChE.At the second stage, we tried to produce specific polyclonal antibody to the catalytic subunit of purified Koi carp AChE, which is used for the development of the new method to assess organophosphate insecticides exposure. Unfortunately, the antibody was not obtained.