The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4.
NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate.
Shaw
P M
PM
Department of Cell Biology, New York University Medical Center 10016.
Reiss
A
A
Adesnik
M
M
Nebert
D W
DW
Schembri
J
J
Jaiswal
A K
AK
D000818:Animals; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D004147:Dioxins / Q000494:pharmacology*; D004790:Enzyme Induction; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D016660:NAD(P)H Dehydrogenase (Quinone); D010957:Plasmids; D011808:Quinone Reductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D004147:Dioxins; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases; D008058:Dihydrolipoamide Dehydrogenase