Immunochemical relationships of cytochrome P4503A-like proteins in teleost fish

Abstract

Multiple P450 proteins have been purified from several teleost species, including rainbow trout (Oncorhynchus mykiss), scup (Stenotomus chrysops) and Atlantic cod (Gadus morhua). Identity, relationships and/or functions have been established in these fish species for the cytochrome P4501 As. Information about the structure, function, regulation and relationships of other piscine cytochrome P450 (CYP) proteins is sparse. In the present study we have focused on constitutively expressed CYP forms, P450con and LMC5 isolated from rainbow trout, P450A from scup, and P450b from Atlantic cod, and we consider evidence for the relationship of these proteins to mammalian members of the CYP3A subfamily. Reciprocal western blot analysis shows that P450con and LMC5, isolated from rainbow trout in two different laboratories, are closely related and ostensibly identical proteins. These trout proteins show specific reciprocal cross-reactivity with scup P450A, and polyclonal antibodies (PAb) to the trout and scup proteins both recognize cod P450b, indicating that rainbow trout P450con/LMC5, scup P450A and cod P450b are immunochemically-related proteins. In analyses of liver microsomes of trout, scup and cod, PAb to trout P450con/LMC5 and scup P450A recognize only bands that are identical in migration to the CYP proteins purified from these species, and which were used as immunogens. These CYP proteins purified from fish are each immunochemically-related to mammalian CYP3A proteins, showing recognition by PAb to human CYP3A4 and to rat CYP3A1. PAb to the mammalian CYP3As also recognize the same bands in liver microsomes from these fish species as seen by PAb to the fish proteins. These results strongly suggest that these fish proteins are members of theCYP3 gene family and probably theCYP3A subfamily. Although sequence analysis is required before their designation in the CYP3A subfamily can be confirmed and specified, we refer to these as CYP3A-like. Immunoblot analyses of hepatic microsomes from other fish species with PAb to scup P450A and trout P450con show that multiple CYP3A-like proteins are expressed in liver of several species, including killifish (Fundulus heteroclitus) and winter flounder (Pleuronectes americanus). Important questions still remain to be addressed concerning CYP3A structure, multiplicity, physiological function, regulation and metabolism of endogenous as well as exogenous substrates in fish.