Purification and Characterization of the Hepatic Microsomal Monooxygenase System from the Coastal Marine Fish 'Stenotomus chrysops'

摘要

Three cytochrome P-450 forms (P-450A, P-450B, and P-450E) were highly purified (8-12 nmol/mg) from liver of untreated marine fish S. chrysops (scup). Scup NADPH-cytochrome P-450 reductase was purified to electrophoretic homogeneity, had a specific activity of 45-60 U/mg with cytochrome c and contained both FAD and FMN. The three P-450 isozymes had distinct spectroscopic properties, substrate profiles, minimum molecular weights (52.7, 45.9 and 54.3 K, respectively), and different peptide maps when digested with a-chymotrypsin or S. aureus V8 protease. A form cytochrome P-450D was also resolved and partially purified. Activity with ethoxycoumarin, acetanilide and alpha-naphthoflavone, and its physico-chemical properties, indicate that P-450E is identical to the major microsomal P-450 induced by 3-MC. Addition of purified scup cytochrome b5 to reconstitions stimulated some catalysis, depending on the P-450 isozyme and substrate. P-450A was generally influenced by the presence of b5, and stimulation was greater with scup than with rabbit liver cytochrome b5. 7-ethoxyresorufin 0-deethylase activity of P-450E was absolutely dependent on scup cytochrome b5.