C. Porte et al., PARTIAL-PURIFICATION AND PROPERTIES OF CYTOCHROME-P450 FROM DIGESTIVEGLAND MICROSOMES OF THE COMMON MUSSEL, MYTILUS-EDULIS-L, Marine environmental research, 39(1-4), 1995, pp. 27-31
Cytochrome P450 from the digestive gland of M. edulis was partially pu
rified by sodium cholate solubilization, 4-15% polyethylene glycol fra
ctionation, and octyl-Sepharose affinity, DEAE-Sephacel ion-exchange a
nd hydroxylapatite chromatography (yields of up to 7-10%). Three peaks
were resolved by DEAE-Sephacel chromatography (termed peaks 1-3). P45
0 specific content was increased from 26 to 800 pmol per mg protein, a
nd the ratio of P450 content to NADPH-cytochrome c (P450) reductase ac
tivity reduced by a factor of 250. Oxidised spectrum lambda max of P45
0 was 410.5+/-1.5 nm. Type II difference spectra were seen with both t
ype II (clotrimazole, metyrapone) and type I (alpha-naphthoflavone, 7-
ethoxy-coumarin) compounds. Western blotting with polyclonal anti-P450
1A 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 c
umene hydroperoxide was indicated to produce diones and protein adduct
s 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, metab
olism in absence of NADPH and added P450 reductase, and predominance o
f diones.