Preparation of highly purified cytochrome P4501A1 from leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular and immunochemical properties

Cytochrome P4501A1 was purified to electrophoretic homogeneity from the liv er microsomes of feral fish leaping mullet (Liza saliens) collected in Izmi r Bay, Aegean coast of Turkey. Purification of cytochrome P4501A1 involved anion exchange chromatography of Emulgen 913-cholate solubilized microsomes on first- and second-DEAE-cellulose columns, hydrophobic interaction chrom atographies of the partially purified cytochrome P4501A1 on Porapak Q and p henyl-Sepharose CL-4B and further purification on adsorption chromatography on the hyrdroxylapatite column. Finally, it is further concentrated and pu rified on the third DEAE-cellulose column. The purified cytochrome P4501A1 was characterized with respect to spectral, electrophoretic, immunochemical and biocatalytic properties. Cytochrome P4501A1, purified 32-fold with a s pecific content of 15-17 nmoles P450 (mg protein)(-1), produced a single ba nd on SDS-polyacrylamide gel electrophoresis having monomer molecular weigh t of 58000 +/- 500. Absolute absorption spectrum of the purified cytochrome P4501A1 fractions showed maximal absorption at 417.5 nm and GO-difference Spectrum of dithionite-reduced cytochrome P4501A1 gave a peak at 448 nm. Pu rified P4501A1 was found to be active in the O-deethylation of 7-ethoxyreso rufin in the reconstituted system containing purified fish liver cytochrome P450 reductase and synthetic lipid. However, it was unable to catalyze the oxidation of the other monooxygenase substrates such as benzphetamine and aniline known to be specific for the other isozymes. Purified L. saliens li ver microsomal cytochrome P4501A1 showed strong cross-reactivity with the a ntibodies directed against the cytochrome P4501A1 homologues purified from other teleost species such as rainbow trout and scup. Spectral, electrophor etic, immunochemical and biocatalytic properties of the purified cytochrome P4501A1 strongly suggested that it is the CYP1A1 in the L. saliens liver. (C) 1998 Elsevier Science Inc. All rights reserved.