Characterization and partial purification of microsomal NAD(P)H : quinone oxidoreductases

Quinone oxidoreductases are flavoproteins that catalyze two-electron reduct ion and detoxification of quinones. This leads to the protection of cells a gainst toxicity, mutagenicity, and cancer due to exposure to environmental and synthetic quinones and its precursors. Two cytosolic forms of quinone o xidoreductases [NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxi doreductase 2 (NQO2)] were previously identified, purified, and cloned. A r ole of cytosolic NQO1 in protection of cells from oxidative stress, cytotox icity, and mutagenicity of quinones was established. Currently, we have cha racterized and partially purified the NQO activity from rat liver microsome s. This activity was designated as microsomal NQO (mNQO). The mNQO activity showed significantly higher affinity for NADH than NADPH as electron donor s and catalyzed reduction of 2,6-dichlorophenolindophenol and menadione. Th e mNQO activity was insensitive to dicoumarol, a potent inhibitor of cytoso lic NQO1, Western analysis of microsomal proteins revealed 29- and 18-kDa b ands that cross-reacted with polyclonal antibodies raised against cytosolic NQO1. The mNQO activity was partially purified by solubilization of micros omes with detergent Chaps, ammonium sulfate fractionation, and DEAE-Sephace l column chromatography. The microsomal mNQO proteins are expected to provi de additional protection after cytosolic NQOs against quinone toxicity and mutagenicity. (C) 2000 Academic Press.