Mouse liver P450Coh: genetic regulation of the pyrazole-inducible enzyme and comparison with other P450 isoenzymes

Abstract

Genetic experiments with two inbred strains of mice, AKR/J and DBA/2N, show a single major gene inheritance of additive mode for pyrazole-inducible coumarin 7-hydroxylase. Intragroup variation in the enzyme activity further suggests the contribution of minor modifying genes to the final enzyme activity. Western blot analysis with a polyclonal antibody raised against the purified isozyme P450Coh (highly active in the 7-hydroxylation of coumarin) showed that a difference in the amounts of P450Coh protein between the D2 and AKR mice is the reason for the differences in the enzyme activity between the two mouse strains. Accordingly, changes at the regulatory level rather than at the structural gene would explain the genetic difference in the activity of coumarin 7-hydroxylase. This hypothesis is further supported by the identical Km values of the basal and induced enzyme. The inducibility of coumarin 7-hydroxylase by phenobarbital (PB) and its genetic regulation have been previously studied by A. W. Wood and colleagues ((1974) Science 185, 612-614; (1979); J. Biol. Chem. 254, 5641-5646 and 5647-5651). Our present experiments show that the regulation is the same for the pyrazole-inducible enzyme. Furthermore the experiments with anti-P450Coh antibody show that the PB- and pyrazole-inducible proteins have the same molecular weight and are immunologically indistinguishable. This suggests that PB and pyrazole may induce the same enzyme. Immunoinhibition of microsomal coumarin 7-hydroxylase is practically 100% for control animals and after pretreatment with pyrazole or PB. This suggests that in each case the same or immunologically closely related proteins are metabolizing coumarin and that the P450Coh may be the only P450 isoenzyme in mouse liver microsomes catalyzing the 7-hydroxylation of coumarin. The N-terminal amino acid sequence of P450Coh was found to be identical with those from Type I and Type II genes of the mouse P45015 alpha family for the first 21 amino acids. With rat PB-inducible P450b the homology is only 33%. Also the immunological properties of P450Coh are different from those of P450b. This may suggest that P450Coh has a closer association to the steroid 15 alpha-hydroxylase gene family than to the P450IIB subfamily of phenobarbital-inducible isoenzymes.