Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse lives

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Abstract
Phenobarbital-induced coumarin 7-hydroxylase is high in DBA/2J and low in C57BL/6N inbred mice; this genetic difference is encoded by the Coh locus on chromosome 7. The aim of this study was to develop an antibody specific for this cytochrome P-450 polymorphism. P-450 fractions, highly specific for phenobarbital-inducible coumarin 7-hydroxylase activity, were purified from DBA/2J and C57BL/6N mouse liver microsomes. Both proteins are 49 kDa [kilodaltons], as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soret peaks of the reduced cytochrome .cntdot. CO complexes are 451 nm. Reconstituted DBA/2J coumarin 7-hydroxylase activity exhibits a Vmax twice as high as, and a Km value 10-fold less than, the reconstituted C57BL/6N activity. Antibodies were raised in rabbit. By Ouchterlony immunodiffusion, both antibodies show 100% cross-reactivity with DBA/2J and C57BL/6N microsomes and purified antigens. Yet, DBA/2J but not C57BL/6N 7-hydroxylase activity is inhibited by the antibody to DBA/2J P-450. Both DBA/2J and C57BL/6N activities are blocked by the antibody to C57BL/6N P-450. Neither antibody has any effect on liver microsomal d-benzphetamine N-demethylase, ethylmorphine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, acetanilide 4-hydroxylase or aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. The DBA/2J protein most specific for phenobarbital-induced coumarin 7-hydroxylation is designated P-450Coh. Anti-(P-450Coh) precipitates a relatively minor 49-kDa protein from detergent-solubilized microsomes and from in vitro translation of poly(A+)-enriched total RNA of phenobarbital-treated DBA/2J mouse liver, whereas the major phenobarbital-induced P-450 proteins exhibit a molecular mass of .apprx. 51 kDa. The immunoprecipitated translation products correspond to a mRNA of 2100 .+-. 100 nucleotides.