Nakajima M, Nagao M, Iwasa M, Monma-Ohtaki J, Maeno Y, Koyama H, Seko-Nakamura Y, Isobe I, Takatori T
Department of Forensic Medicine, Postgraduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Toxicology. 2000 Nov 23;154(1-3):55-66. doi: 10.1016/s0300-483x(00)00299-7.
To establish a paraquat-resistant Wistar rat strain, we carried out continuous sister-brother mating among rats that survived high-dose intraperitoneal administration of paraquat dichloride (360 mg/kg). The percentages of paraquat-resistant rats among wild rats and among the fifth-generations were 7.1% and 20.6%, respectively. After high-dose paraquat administration, the serum paraquat concentration in sensitive rats was much higher than that in paraquat-resistant rats. The cytosol fraction of liver from paraquat-resistant rats had higher paraquat- and diquat-metabolizing activities than that of liver from paraquat-sensitive rats. By contrast, microsomal fractions from livers of paraquat-resistant and paraquat-sensitive rats had no paraquat- or diquat-metabolizing activity. This paraquat/diquat-metabolizing enzyme was partially purified from paraquat-resistant rat liver cytosol using affinity chromatography for diquat. At the end of the purification procedure, rat liver diquat-metabolizing enzyme was purified 1154-fold to a final specific activity of 32.32 mol/h/mg protein, and an overall recovery of about 0.46% was obtained. This enzyme oxidized diquat to diquat-dipyridone during overnight incubation at 37 degrees C, but only metabolized traces of paraquat. The molecular mass of the enzyme was estimated as 190 kDa, and its isoelectric point of it was 4.6-4.7. Kinetic study revealed the values of K(m) and V(max) to be 35.0 micromol/l and 0.81 micromol/h/ml, respectively.
为建立对百草枯具有抗性的Wistar大鼠品系，我们对经腹腔注射高剂量二氯百草枯（360 mg/kg）后存活的大鼠进行连续的同胞兄妹交配。野生大鼠和第五代大鼠中抗百草枯大鼠的比例分别为7.1%和20.6%。高剂量百草枯给药后，敏感大鼠血清中的百草枯浓度远高于抗百草枯大鼠。抗百草枯大鼠肝脏的胞质部分比百草枯敏感大鼠肝脏具有更高的百草枯和敌草快代谢活性。相比之下，抗百草枯大鼠和百草枯敏感大鼠肝脏的微粒体部分均无百草枯或敌草快代谢活性。利用敌草快亲和色谱法从抗百草枯大鼠肝脏胞质中部分纯化了这种百草枯/敌草快代谢酶。在纯化过程结束时，大鼠肝脏敌草快代谢酶纯化了1154倍，最终比活性为32.32 μmol/h/mg蛋白，总回收率约为0.46%。该酶在37℃过夜孵育期间将敌草快氧化为敌草快二吡啶酮，但仅代谢痕量的百草枯。该酶的分子量估计为190 kDa，其等电点为4.6 - 4.7。动力学研究表明，K(m)和V(max)值分别为35.0 μmol/L和0.81 μmol/h/ml。
