Characterization and partial purification of microsomal NAD(P)H:quinone oxidoreductases.
Quinone oxidoreductases are flavoproteins that catalyze two-electron reduction and detoxification of quinones. This leads to the protection of cells against toxicity, mutagenicity, and cancer due to exposure to environmental and synthetic quinones and its precursors. Two cytosolic forms of quinone oxidoreductases [NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2)] were previously identified, purified, and cloned. A role of cytosolic NQO1 in protection of cells from oxidative stress, cytotoxicity, and mutagenicity of quinones was established. Currently, we have characterized and partially purified the NQO activity from rat liver microsomes. This activity was designated as microsomal NQO (mNQO). The mNQO activity showed significantly higher affinity for NADH than NADPH as electron donors and catalyzed reduction of 2,6-dichlorophenolindophenol and menadione. The mNQO activity was insensitive to dicoumarol, a potent inhibitor of cytosolic NQO1. Western analysis of microsomal proteins revealed 29- and 18-kDa bands that cross-reacted with polyclonal antibodies raised against cytosolic NQO1. The mNQO activity was partially purified by solubilization of microsomes with detergent Chaps, ammonium sulfate fractionation, and DEAE-Sephacel column chromatography. The microsomal mNQO proteins are expected to provide additional protection after cytosolic NQOs against quinone toxicity and mutagenicity.
Jaiswal
A K
AK
Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA. ajaiswal@bcm.tmc.edu
D015086:2,6-Dichloroindophenol / Q000378:metabolism; D000818:Animals; D002793:Cholic Acids / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D003902:Detergents / Q000737:chemistry; D001728:Dicumarol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005590:Fractional Precipitation; D007106:Immune Sera / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014812:Vitamin K / Q000378:metabolism; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology
D002793:Cholic Acids; D003902:Detergents; D004791:Enzyme Inhibitors; D007106:Immune Sera; D007527:Isoenzymes; D013439:Sulfhydryl Reagents; D009243:NAD; D014812:Vitamin K; D009249:NADP; D020245:p-Chloromercuribenzoic Acid; D001728:Dicumarol; D015086:2,6-Dichloroindophenol; D016660:NAD(P)H Dehydrogenase (Quinone); C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate