A continuous spectrophotometric determination of hepatic microsomal azo reductase activity and its dependence on cytochrome P-450.
1. A continuous spectrophotometric determination of rat hepatic microsomal anaerobic azo reductase activity has been developed. 2. The addition of soluble flavins (riboflavin, FMN or FAD) greatly increased this NADPH-dependent activity towards a number of azo substrates. 3. Investigations with amaranth as substrate gave an apparent Km of 34 microM and Vmax. of 4 nmol/min per mg of microsomal protein. The inclusion of a fixed concentration of FMN increased Vmax. and greatly decreased Km, the magnitude of these changes reflecting the concentration of flavin present. 4. Investigations using a fixed amaranth concentration over a range of flavin concentrations gave biphasic double-reciprocal plots with two apparent Km and Vmax. values. 5. Pretreatment of animals with cobaltous chloride, 2-allyl-2-isopropylacetamide, carbon tetrachloride, phenobarbitone and 3-methylcholanthrene altered azo reductase activity in parallel with changes in cytochrome P-450 content. 6. The significance of these results is discussed in terms of the electron-transfer components present in the hepatic microsomal fraction.
D000548:Amaranth Dye / Q000378:metabolism; D000818:Animals; D001391:Azo Compounds / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004579:Electron Transport / Q000187:drug effects; D005486:Flavin Mononucleotide / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009601:Nitroreductases; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry
D001391:Azo Compounds; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D000548:Amaranth Dye; D009247:NADH, NADPH Oxidoreductases; D009601:Nitroreductases; C021106:azoreductase