PARTIAL-PURIFICATION AND PROPERTIES OF CYTOCHROME-P450 FROM DIGESTIVE GLAND MICROSOMES OF THE COMMON MUSSEL, MYTILUS-EDULIS-L

Cytochrome P450 from the digestive gland of M. edulis was partially purified by sodium cholate solubilization, 4-15% polyethylene glycol fractionation, and octyl-Sepharose affinity, DEAE-Sephacel ion-exchange and hydroxylapatite chromatography (yields of up to 7-10%). Three peaks were resolved by DEAE-Sephacel chromatography (termed peaks 1-3). P450 specific content was increased from 26 to 800 pmol per mg protein, and the ratio of P450 content to NADPH-cytochrome c (P450) reductase activity reduced by a factor of 250. Oxidised spectrum lambda max of P450 was 410.5+/-1.5 nm. Type II difference spectra were seen with both type II (clotrimazole, metyrapone) and type I (alpha-naphthoflavone, 7-ethoxy-coumarin) compounds. Western blotting with polyclonal anti-P4501A from perch (Perca fluviatilis) gave a single band of approximately 54 kDa molecular weight. A reconstituted system containing peak 2 or 3, rat liver P450 reductase, lipid and NADPH metabolised benzo[a]pyrene to diones, diols, phenols and putative protein adducts. Peak 2 plus cumene hydroperoxide was indicated to produce diones and protein adducts only. Peak 2 alone was indicated to produce diones and phenols. The major free metabolites in all cases were diones (53-100%). The results indicate the existence of a P4501A-like enzyme in M. edulis, possibly with unusual properties as indicated by the difference spectra, metabolism in absence of NADPH and added P450 reductase, and predominance of diones.