Mechanism of azoreduction of dimethylaminoazobenzene by rat liver NADPH-cytochrome P-450 reductase and partially purified cytochrome P-450. Oxygen and carbon monoxide sensitivity and stimulation by FAD and FMN.
We have reported that the hepatocarcinogen dimethylaminoazobenzene (DAB) is reduced by rat liver microsomes in an oxygen- and carbon monoxide-insensitive manner and that activity is induced by clofibrate but no other recognized inducers of cytochrome P-450 activity. In the present study we have shown that the reaction proceeds in a partially purified reconstituted cytochrome P-450 system as well as with purified NADPH-cytochrome P-450 reductase alone. In the latter system, activity is totally inhibited in air whereas the former system is active in air as well as in a carbon monoxide atmosphere. Although clofibrate induces both DAB azoreductase and laurate hydroxylase activities, the suicide substrate 10-undecynoic acid blocks the latter but not the former, implying catalysis by distinct enzymes. FAD and FMN stimulate DAB azoreduction 40-50-fold by both NADPH-cytochrome P-450 reductase alone and by the reconstituted cytochrome P-450 system. However, it was shown that these flavins facilitate electron flow to DAB only from reductase and not from cytochrome P-450. The fact that the reconstituted system, which contains NADPH-cytochrome P-450 reductase, is oxygen insensitive suggests that there is an obligatory electron flow through cytochrome P-450 to DAB, bypassing the oxygen-sensitive step.
D000818:Animals; D001391:Azo Compounds / Q000378:metabolism*; D002248:Carbon Monoxide / Q000494:pharmacology*; D002994:Clofibrate / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004032:Diet; D005486:Flavin Mononucleotide / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D004124:p-Dimethylaminoazobenzene / Q000378:metabolism*
D001391:Azo Compounds; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D004124:p-Dimethylaminoazobenzene; D009251:NADPH-Ferrihemoprotein Reductase; D002994:Clofibrate; D010100:Oxygen