IMMUNOCHEMICAL RELATIONSHIPS OF CYTOCHROME P4503A-LIKE PROTEINS IN TELEOST FISH

Multiple P450 proteins have been purified from several teleost species , including rainbow trout (Oncorhynchus mykiss), scup (Stenotomus chry sops) and Atlantic cod (Gadus morhua). Identity, relationships and/or functions have been established in these fish species for the cytochro me P4501As. Information about the structure, function, regulation and relationships of other piscine cytochrome P450 (CYP) proteins is spars e. In the present study we have focused on constitutively expressed CY P forms, P450con and LMC5 isolated from rainbow trout, P450A from scup , and P450b from Atlantic cod, and we consider evidence for the relati onship 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 antibodie s (PAb) to the trout and scup proteins both recognize cod P450b, indic ating that rainbow trout P450con/LMC5, scup P450A and cod P450b are im munochemically-related proteins. In analyses of liver microsomes of tr out, scup and cod, PAb to trout P450con/LMC5 and scup P450A recognize only bands that are identical in migration to the CYP proteins purifie d from these species, and which were used as immunogens. These CYP pro teins purified from fish are each immunochemically-related to mammalia n CYP3A proteins, showing recognition by PAb to human CYP3A4 and to ra t 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 p roteins. These results strongly suggest that these fish proteins are m embers of the CYP3 gene family and probably the CYP3A subfamily. Altho ugh sequence analysis is required before their designation in the CYP3 A subfamily can be confirmed and specified, we refer to these as CYP3A -like. Immunoblot analyses of hepatic microsomes from other fish speci es with PAb to scup P450A and trout P450con show that multiple CYP3A-l ike proteins are expressed in liver of several species, including kill ifish (Fundulus heteroclitus) and winter flounder (Pleuronectes americ anus). Important questions still remain to be addressed concerning CYP 3A structure, multiplicity, physiological function, regulation and met abolism of endogenous as well as exogenous substrates in fish.