Development of a new method to assess organophosphate insecticides exposure

自several chemicals such as organophosphate and carbamate insecticides and heavy metals inhibitcholinesterases,the tissue levels of acetylcholinesterase(AChE) in various fishes have been used as a potentialbiomarker for exposure to these chemicals in aquatic environments. in order to make AChE a more可靠且有用的生物化学家it seems essential that a new method to assess organophosphate insecticides exposure这是不受限制的，不同的差异在tissue等级的AChE，be developed. It is so desirable that the AChE is characterized well after purification，自catalytic properties and inhibitor sensitivities of the enzyme to various chemicals vary fromspecies 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)，the fishes used for environmental monitoring in aquatic environmentand tried to develop the new method described . at the第一步，我们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> proteinand the molecular mass of the catalytic subunit was 6.8kDa. the characterizations of the purifiedenzyme in catalytic property，substrate specificity and inhibitor specificity demonstrate that the purified AChE from Koi carpmuscle is certainly an ach . at the second stage，我们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 insecticidesexposure. Unfortunately, the antibody not obtained。

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.