Characterization of carbonyl reducing activity in continuous cell lines of human and rodent origin.
Carbonyl reduction was investigated in the continuous cell lines V79, NCI-H322 and C2REV7 by using the ketone compound metyrapone as a substrate. Metyrapone reducing enzymes were characterized by evaluating the cosubstrate requirement and by testing the sensitivity of this reaction to specific inhibitors. All cell lines were found to produce metyrapol at a linear rate over a time course of at least 48 hr, when tested in cultured monolayers. In general, cytosolic metyrapone reduction exceeds microsomal activity several-fold in all three cell lines. Quercitrin turned out to be the strongest inhibitor in all fractions, except in NCI-H322 microsomes where it had no effect. Consequently, carbonyl reductase is suspected to be responsible for metyrapone reduction in the cytosol and microsomes of V79 and C2REV7 cells as well as in the cytosol of NCI-H322 cells. Simultaneous sensitivity towards quercitrin, dicoumarol, indomethacin and 5 alpha-dihydrotestosterone in some cases points to the existence of different isozymes of carbonyl reductase. In NCI-H322 microsomes only dicoumarol and indomethacin decrease metyrapol formation, thus pointing to an isozyme of NAD(P)H:quinone-oxidoreductase. Concerning cosubstrate requirements metyrapone reducing enzymes show a strong preference for NADPH, thus confirming the involvement of carbonyl reductase in this reaction. In conclusion, carbonyl reduction of metyrapone in continuous cell lines is mediated by carbonyl reductases due to the common sensitivity towards the diagnostic inhibitor quercitrin and due to the strong preference for NADPH as cosubstrate. According to its maintenance in permanent cell lines carbonyl reductase seems to be an essential and constitutive enzyme, which probably fills an important role in normal cell physiology.
Gebel
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T
Department of Pharmacology and Toxicology, School of Medicine, University of Marburg, Federal Republic of Germany.
Maser
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E
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D002460:Cell Line / Q000201:enzymology; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D008797:Metyrapone / Q000031:analogs & derivatives / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011794:Quercetin / Q000031:analogs & derivatives / Q000494:pharmacology; D012377:Rodentia; D013347:Subcellular Fractions / Q000378:metabolism
C002196:metyrapol; C012526:quercitrin; D009249:NADP; D011794:Quercetin; D000429:Alcohol Oxidoreductases; D008797:Metyrapone