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 continu ous sister-brother mating among rats that survived high-dose intraperitonea l administration of paraquat dichloride (360 mg/kg). The percentages of par aquat-resistant rats among wild rats and among the fifth-generations were 7 .1% and 20.6%, respectively. After high-dose paraquat administration, the s erum paraquat concentration in sensitive rats was much higher than that in paraquat-resistant rats. The cytosol fraction of liver from paraquat-resist ant rats had higher paraquat- and diquat-metabolizing activities than that of liver from paraquat-sensitive rats. By contrast, microsomal fractions fr om livers of paraquat-resistant and paraquat-sensitive rats had no paraquat - or diquat-metabolizing activity. This paraquat/diquat-metabolizing enzyme was partially purified from paraqu at-resistant rat liver cytosol using affinity chromatography for diquat. At the end of the purification procedure, rat liver diquat-metabolizing enzym e was purified 1154-fold to a final specific activity of 32.32 mol/h/mg pro tein, and an overall recovery of about 0.46% was obtained. This enzyme oxid ized diquat to diquat-dipyridone during overnight incubation at 37 degreesC , 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. Kin etic study revealed the values of K-m and V-max to be 35.0 mu mol/l and 0.8 1 mu mol/h/ml, respectively. (C) 2000 Elsevier Science Ireland Ltd. All rig hts reserved.