Isolation of a novel hepatic CYP2-related protein from channel catfish, Ictalurus punctatus

Channel catfish (Ictalurus punctatus) have been previously shown to express two major cytochrome P450 (CYP) protein bands that are cross-reactive with anti-CYP2K1 (rainbow trout, Oncorhynchus mykiss) antibodies on Western blo ts. These proteins appear to be the major constitutive CYPs in channel catf ish and show distinct sex- and age-specific variations in expression. Becau se I. punctatus is an important agricultural and ecological commodity, and because it displays a high degree of resistance to the toxic effects of man y pesticides, the molecular and catalytic characteristics of its biotransfo rmation systems are of interest to those in areas of environmental science and aquaculture research. Using a chromatographic method similar to that em ployed in the purification of other fish CYP2 enzymes, a single CYP2-relate d protein (CM-HA3) was isolated from channel catfish hepatic microsomes. Th e isolated protein displays a relative molecular mass of approximately 47 k Da, and a CO-reduced difference spectrum lambda(max) of 449.6 nm. The seque nce of 15 residues at the amino-terminal of CM-HA3 is 27% identical to both CYP2K1 and CYP2M1 isoforms of rainbow trout. Correlational analysis was em ployed to characterize potential substrates for this isoform, but no signif icant relationship was observed with E-2 hydroxylation, testosterone hydrox ylation, or 7-ethoxycoumarin O-deethylase activities. These data indicate t hat CM-HA3 is a CYP2 family protein, with as yet uncharacterized substrate specificities.