Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450 isozyme 3a.
The hydroxylation of p-nitrophenol to 4-nitrocatechol was investigated using rabbit hepatic microsomes and six purified isozymes of cytochrome P-450. The microsomal activity was maximal at pH 6.8 and at 100 microM p-nitrophenol. At higher substrate concentrations inhibition was observed. At pH 6.8 and 100 microM p-nitrophenol, isozyme 3a exhibited the highest activity of the purified isozymes: 3.4-fold more active than isozyme 6, and 8-fold more active than isozymes 2 and 4. The isozyme 3a-catalyzed hydroxylation reaction was stimulated 2.4-fold by the addition of a 4:1 ratio of cytochrome b5/P-450. At optimal concentrations of cytochrome b5, isozyme 3a was 8- to 9-fold more active than isozymes 2 and 6 and 20-fold more active than isozyme 4. Under the same conditions, isozyme 3a-catalyzed butanol oxidation was inhibited 40%. Antibodies to isozyme 3a inhibited greater than 95% of the p-nitrophenol hydroxylase activity of microsomes from untreated or from ethanol- or acetone-treated rabbits. The microsomal hydroxylase activity was linearly correlated with the microsomal concentration of isozyme 3a (correlation coefficient of 0.94) and had an intercept near zero. The results from reconstitution, antibody inhibition, and correlation experiments indicate that isozyme 3a is the principal catalyst of rabbit microsomal p-nitrophenol hydroxylation. The ability of the ethanol-inducible isozyme to catalyze catechol formation may be important in the ethanol-enhanced toxicity of aromatic compounds such as benzene.
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D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism*; D003573:Cytochrome b Group / Q000378:metabolism; D015786:Cytochromes b5; D004790:Enzyme Induction; D000431:Ethanol / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D009596:Nitrophenols / Q000378:metabolism*; D011817:Rabbits
D003573:Cytochrome b Group; D007527:Isoenzymes; D009596:Nitrophenols; D000431:Ethanol; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; C024836:4-nitrophenol