RAT-LIVER CYTOCHROME-P450 2B3 - STRUCTURE OF THE CYP2B3 GENE AND IMMUNOLOGICAL IDENTIFICATION OF A CONSTITUTIVE P450 2B3-LIKE PROTEIN IN RAT-LIVER

The cytochrome P450 2B subfamily in the rat contains an estimated eigh t to eleven members at the genomic level. Synthesis in the liver of th e prototypic forms P450 2B1 and P450 2B2 is dramatically induced by ph enobarbital. The 1.9-kb mRNA for P450 2B3, a third member of the P450 2B subfamily, is constitutively present in rat liver but is not induci ble by phenobarbital. We have now cloned and sequenced exonic sequence s corresponding to the entire 2B3 mRNA and determined their exon-intro n structure, which is identical to that of CYP2B1/CYP2B2 and other CYP 2B genes. A putative CYP2B3 transcription start site was identified an d CYP2B3 5'- and 3'-flanking sequences were compared to those of CYP2B 1 and CYP2B2. CYP2B3, like CYP2B1 and CYP2B2, has a modified TATA box preceding the transcription start site and lacks the canonical polyade nylation signal preceding the poly(A) site. A 2B3 expression vector, p MT2-2B3, directed the synthesis in COS-1 cells of an similar to 50-kD protein detectable on Western blots with a polyclonal antibody and wit h one of four monoclonal antibodies raised against 2B1 but not with a polyclonal antibody raised against P450 PB6. The 2B3 protein migrated with a slightly higher electrophoretic mobility than 2B1 acid comigrat ed with a protein detected by anti-2B1 antibodies in liver microsomes from untreated rats. The results indicate that a 2B3-like protein is p resent in rat liver and that it is distinct from P450 PB6 and other kn own constitutive rat hepatic P450s.