Investigating the CYP2B1-WM Variant: A Case of "Natural" Site-Directed Mutagenesis
Strom, Scott R.B.
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The phenobarbital-inducible cytochrome P4S0 2Bl (CYP2Bl) and 2B2 (CYP2B2) isozymes bear identical structure apart from 13 amino acid differences in the carboxyl-terminal half of the protein. In this study, comparison is made between liver microsomes isolated from inbred Fischer and Munich-Wistar rats, treated with phenobarbital and untreated, in order to assess the latter's efficacy in carrying out activities specific to the CYF2B subfamily. The Munich-Wistar rat bears a deletion of the CYP2B2 gene (Omiecinski et al., 1992), and an amino acid substitution (Gly 478 -> Ala) in CYP2Bl which increases the protein's structural homology to CYP2B2 (yielding allele CYP2BI-WM) (Kedzie et aI., 1991). There was a greater increase in cytochrome P450 content (nmol P450/min/mg microsomal protein) after phenobarbital-induction in Munich-Wistar rats. 7-Pentoxy- and 7-benzyloxyresorufin have been found to be specific substrates for the P450 2B subfamily (Nerurkar et al., 1993); Odealkylation activities exhibited by Fischer microsomes were twice those shown by Munich-Wistar microsomes. SDS-PAGE/Western immunoblot analysis confirmed the presence of CYP2Bl and CYP2B2 in Fischer microsomes, whereas Munich-Wistar microsomes were shown to contain only CYP2Bl. Formation of the metabolite methylhydroxylidocaine (Me-OH-Lid), shown to be CYP2B2- dependent (Oda et ai., 1989), was assayed by incubation of each microsome type with lidocaine. Despite the absence of CYP2B2 in Munich-Wistar microsomes, formation of Me-OH-Lid was observed, though at a lower activity than observed with Fischer microsomes. From this, it can be concluded that the amino acid substitution in CYP2BI-WM is important in conferring upon it the activity of the CYP2B2 isozyme.