Graduate Thesis Or Dissertation
 

Cloning, sequencing and aflatoxin B₁ metabolism by multiple rainbow trout CYP1A cDNAs expressed in yeast

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  • Previous reports indicate the presence of multiple CYP1A sequences in rainbow trout, but their functional differences are unknown. This report describes the cloning and partial characterization of four trout CYP1A cDNAs, which are given the tentative designations CYP1A1v2, v3, v4, and v5. Comparison among these four and three previously reported trout CYP1A sequences reveals that all of the nucleotide and translated amino acid sequences all are closely related (96.9-99.4% cDNA identity; 95.2-99.4% amino acid sequence identity) but none are identical. Six of these sequences encode proteins of 522 amino acids, and one encodes a protein of 536 amino acids. Expression vectors containing the cDNAs for CYP1A1v2, v3, and v4 were transformed into yeast, yielding microsomal hemoprotein CYP contents (63, 156, 96 pmol/mg) comparable to those reported for human CYP1A1 (68-156 pmol/mg) expressed in this system (Eugster et al., 1990, Biochem. Biophys. Res. Commun. 737-744). Kinetic analysis of CYP1A1v2 and v3 proteins indicated similar but not identical Michaelis constants (20±3 vs 13±2 μM) and molar activities (508±47 vs 218±19 pmol/min/nmol P450) for oxidation of aflatoxin B₁ (AFB₁) to aflatoxin M a reaction characteristic of human CYP1A2. Trout CYP1A1v2 and v3 exhibited lower activity for production of AFB,-8,9-exo-epoxide, also a human CYP1A2 activity. Kinetic data for ethoxyresorufin 0deethylation, a prototypical mammalian CYP1A1 activity, also revealed modest but distinct differences in which CYP1A1v3 was more active for this substrate (Km=0.07 ± 0.01 μM, Vm=1398 ± 95 pmol/min/nmol P450) than was CYP1A1v2 (Km=0.15 ± 0.03 μM, Vm=684 ± 83 pmol/min/nmol p450). Interestingly, CYP1A1v4 showed no catalytic activity towards AFB ethoxyresorufin, or 7,12-dimethylbenzanthracene despite formation of a hemoprotein. These results together with previous studies demonstrate the presence in various rainbow trout populations of at least seven CYPIA cDNAs representing gene duplication or allelic variation. Present results show that one of three such cDNA sequences encodes a CYP1A hemoprotein with no apparent catalytic activity, that two of the encoded proteins possess certain catalytic properties common to both human CYP1A1 and CYP1A2, and that the sequence differences, though small, are reflected in enzymic properties that can be distinguished.
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