Uroporphyrinogen oxidation catalyzed by reconstituted cytochrome P450IA2.
Previous work suggested that the oxidation of uroporphyrinogen to uroporphyrin is catalyzed by cytochrome P450IA2. Here we determined whether purified reconstituted mouse P450IA1 and IA2 oxidize uroporphyrinogen. Cytochromes P450IA1 and IA2 were purified from hepatic microsomes from 3-methylcholanthrene (MC)-treated C57BL/6 mice, using a combination of affinity chromatography and high performance liquid chromatography. Reconstituted P450IA1 was more active than P450IA2 in catalyzing ethoxyresorufin-O-deethylase (EROD) activity, whereas P450IA2 was more active than P450IA1 in catalyzing uroporphyrinogen oxidation (UROX). Both reactions required NADPH, NADPH-cytochrome P450 reductase, and either P450IA1 or IA2. Ketoconazole competitively inhibited both EROD and UROX activities, in microsomes from MC-treated mice. Ketoconazole also inhibited UROX catalyzed by reconstituted P450IA2. In contrast, ketoconazole did not inhibit UROX catalyzed by xanthine oxidase in the presence of iron-EDTA. Superoxide dismutase, catalase, and mannitol inhibited UROX catalyzed by xanthine oxidase/iron-EDTA, but did not affect UROX catalyzed by either microsomes or reconstituted P450IA2. These results suggest that UROX catalyzed by P450IA2 in microsomes and reconstituted systems does not involve free reactive oxygen species. Two known substrates of cytochrome P450IA2, 2-amino-3,4-dimethylimidazole[4,5-f]quinoline and phenacetin, were shown to inhibit the microsomal UROX reaction, suggesting that uroporphyrinogen binds to a substrate-binding site on the cytochrome P450.
Lambrecht
R W
RW
Veterans Administration, White River Junction, Vermont 05009.
Sinclair
P R
PR
Gorman
N
N
Sinclair
J F
JF
D000818:Animals; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D010615:Phenacetin / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D014577:Uroporphyrinogens / Q000378:metabolism*
D011804:Quinolines; D014577:Uroporphyrinogens; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; D010615:Phenacetin; C036989:2-amino-3,4-dimethylimidazo(4,5-f)quinoline; D007654:Ketoconazole