Mass spectrometric analyses of microsomal cytochrome P450 isozymes isolated from β-naphthoflavone-treated Atlantic cod (Gadus morhua) liver reveal insights into the cod CYPome

Abstract

Four cytochrome P450 (CYP) isozymes were purified earlier from liver microsomes of Atlantic cod (Gadus morhua) exposed to β-naphthoflavone. These isozymes were named P-450a-d and described with regard to optical properties, enzymatic activity, molecular weight and immunological reactivity. Subsequent analyses of the individual CYP fractions have shown that P-450c corresponds to a CYP1A isozyme, and immunochemical analyses have indicated that P-450b belongs to the CYP3A subfamily. However, no sequence data has been obtained to confirm these results, and the identities of the P-450a and P-450d have also remained unknown. The sequencing effort of the cod genome and expanding cod EST-databases (www.codgenome.no) have substantially increased the number of protein sequences available from cod. Mass spectrometric techniques were therefore applied to further characterize the proteins in historically archived samples of P-450a-d fractions. These analyses revealed large heterogeneities of CYPs within the purified samples. The most prominent CYP isozymes present include members of the CYP1A, CYP1C, CYP3A and CYP4V families. In total, 29 unique CYPs belonging to 9 CYP gene families and 15 subfamilies were identified with mass spectrometry. This analysis was also accompanied with genomic mining as the first step to unveil the full suite of cod CYP genes. In total, 55 CYP genes were predicted from the cod genome, distributed among 16 CYP gene families that are also present in other fish as well as mammals. Importantly, the majority of the CYPs revealed in this study have not previously been reported from cod, and represents the first proteomic survey to uncover the expressed complement of CYPs in any non-mammalian species.