Purification and characterization of diquat (1,1'-ethylene-2,2'-dipyridylium)- metabolizing enzyme from paraquat-resistant rat liver cytosol

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°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 μmol/l and 0.81 μmol/h/ml, respectively. (C) 2000 Elsevier Science Ireland Ltd.