PURIFICATION AND CHARACTERIZATION OF 3 CONSTITUTIVE CYTOCHROME-P-450 ISOFORMS FROM BOVINE OLFACTORY EPITHELIUM

Three constitutive forms of cytochrome P-450 (P-450s) were isolated fr om olfactory microsomes of cattle. The purified P-450s, designated P-4 50bov1, P-450bov2 and P-450bov3, were electrophoretically nearly homog eneous by SDS/PAGE and their apparent relative molecular masses were e stimated to be 50000, 53000 and 51000 respectively. As indicated by se veral criteria including the N-terminal sequence and absorption spectr a, the three olfactory forms of P-450 were distinct from each other an d from all the other P-450s currently known in cattle. P-450bov1 and P -450bov2 were purified in the low-spin state, whereas P-450bov3 was in the high-spin state. Studies to evaluate, by Western blot analysis, t he reactivity of these purified P-450s with antibodies raised against rat hepatic P-450 2E1, 2B, 1A and 3A and rabbit olfactory P-450NMa and P-450NMb showed that P-450bov3 strongly cross-reacted with anti-P-450 NMb IgG, and P-450bov1 moderately with anti-P-450NMa IgG. As determine d by immunoblots, P-450bov1 and P-450bov3 represented a great portion of the total olfactory P-450. In a reconstituted system with NADPH:cyt ochrome P-450 reductase and phospholipids, P-450bov1 was more active i n the metabolism of xenobiotic compounds (i.e. O-de-ethylation of etho xycoumarin and N-demethylation of hexamethylphosphoramide) than toward s endogenous substrates (testosterone and progesterone). Conversely, P -450bov3 metabolized the xenobiotics at lower rates but exhibited tota l oxidation rates of the above sex hormones higher than those of P-450 bov1. From the comparison of the catalytic, immunochemical and structu ral properties, it was inferred that P-450bov1 and P-450bov3 are the b ovine orthologues of P-450NMa (2A) and P-450NMb (2G1) respectively, th e only two olfactory P-450s previously purified from rabbit. P-450bov2 , which showed low activity toward some exogenous and endogenous compo unds, represents a novel purified olfactory hemoprotein possibly belon ging to the 3A subfamily. These results are consistent with a specific presence of catalytically and structurally similar P-450s, at least f or the major ones, in the olfactory mucosa of mammals.