Structural gene products of the Ah locus. Genetic and immunochemical evidence for two forms of mouse liver cytochrome P-450 induced by 3-methylcholanthrene.

With the use of heterogenic stock (HS) mice, more than g-fold differences in hepatic microsomal 3-methylcholanthrene-inducible aryl hydrocarbon (benzo[alpyrene) hydroxylase (EC 1.14.14.2) activity and at least 4-fold differences in 3-methylcholanthrene-inducible acetanilide 4-hydroxylase activity were found. These data strongly suggest independent genetic control of two inducible cytochrome P-450-mediated monooxygenase activities known to be associated with the murine Ah locus. The procedure is described for isolating two inducible forms of partially separated P-450 from C57BL/6N treated with 3-methylcholanthrene, and an antibody to each of these forms was developed. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a molecular weight of about 55,000 is estimated for both partially separated hemoproteins. Limited proteolysis reveals differences in the peptide composition of these two cytochromes. PI-450 is more closely associated with polycyclic aromatic compound-inducible aryl hydrocarbon hydroxylase activity and NADPH oxidation in the presence of benzo[a]pyrene, whereas P-448 is more closely associated with polycyclic aromatic compoundinducible acetanilide 4-hydroxylase activity and NADPH oxidation in the presence of acetanilide. The Soret peaks of the reduced hemoprotein*CO complex are about 449.3 and 448.0 nm for PI-450 and P-448, respectively. Goat anti-PI-450 immunoglobulin inhibits 3-methylcholanthrene-inducible aryl hydrocarbon hydroxylase activity while not affecting 3-methylcholanthrene-inducible acetanilide 4-hydroxylase activity. Rabbit anti-P-448 immunoglobulin blocks 3-methylcholanthrene-inducible acetanilide 4-hydroxylase activity while not affecting 3-methylcholanthrene-inducible aryl hydrocarbon hydroxylase activity. Evidence is also presented for the possible presence of P-448 in the liver microsomes of the control, untreated mouse. By means of the pyridoxal phosphate/NaB[:‘H& method for labeling intact microsomes in vitro followed by immunoprecipitation, it was confirmed that transplacental PI-450 induction (and its associated aryl hydrocarbon hydroxylase activity) in fetal mouse liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin develops earlier in gestational age than P-448 induction (and its associated acetanilide 4-hydroxylase activity) by 2,3,7,8-tetrachlorodibenzo-p-dioxin. These data, plus studies involving inhibitors in vitro,