Further immunochemical and biocatalytic characterization of CYP1A1 from feral leaping mullet liver (Liza saliens) microsomes

CYP1A is known to play important roles in the metabolism, detoxification an d bioactivation of carcinogens and other xenobiotics in animals including f ish. In our laboratory, CYP1A1 was obtained in a highly purified form with a specific content of 15-17 nmol P450 per mg protein from liver microsomes of feral fish, leaping mullet (Liza saliens). Purified mullet CYP1A1 showed a very high substrate specificities for 7-ethoxyresorufin and 7-methoxyres orufin in a reconstituted system containing purified fish P450 reductase an d lipid. In addition, effects of each individual components of the reconsti tuted system, i.e., CYP1A1 and P450 reductase on 7-methoxyresorufin O-demet hylase (MROD) activity were studied. 7-ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by cl-naphthoflavone (ANF). At 0.5 and 2.5 mu M, ANF inhibited EROD activity by 90 and 98%, respectively. Mullet CYP1A 1 did not catalyze monooxygenations of other substrates such as aniline, et hylmorphine. N-nitrosodimethylamine and p-nitrophenol. Antibodies produced against CYP1A1 orthologues in fish such as trout and scup showed strong cro ss-reactivity with the purified mullet CYP1A1. In addition, anti-L. saliens liver CYP1A1 produced in our laboratory inhibited both the EROD and MROD a ctivities catalyzed by L. saliens liver microsomes but stronger inhibition was observed with EROD activity. On the other hand, anti-mullet CYP1A1 anti bodies showed very weak cross-reactivity with two proteins (presumably CYP1 A1 and CYP1A2) in 3MC-treated rat liver microsomes. Moreover, 3MC-treated r at liver microsomal EROD activity was weakly inhibited by the anti-L. salie ns liver CYP1A1. These results strongly suggested that the purified mullet CYP1A1 is structurally, functionally and immunochemically similar to the CY P1A1 homologues purified from other teleost species but functionally and im munochemically distinct from mammalian CYP1A1. (C) 2000 Elsevier Science In c. All rights reserved.