Akademik Veri Yönetim Sistemi
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY, cilt.121, ss.249-265, 1998 (SCI-Expanded)
Cytochrome P4501A1 was purified to electrophoretic homogeneity from the liver microsomes of feral fish leaping mullet (Liza saliens) collected in Izmir 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 chromatographies of the partially purified cytochrome P4501A1 on Porapak Q and phenyl-Sepharose CL-4B and further purification on adsorption chromatography on the hyrdroxylapatite column. Finally, it is further concentrated and purified 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 specific content of 15-17 nmoles P450 (mg protein)(-1), produced a single band on SDS-polyacrylamide gel electrophoresis having monomer molecular weight 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. Purified P4501A1 was found to be active in the O-deethylation of 7-ethoxyresorufin 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 liver microsomal cytochrome P4501A1 showed strong cross-reactivity with the antibodies directed against the cytochrome P4501A1 homologues purified from other teleost species such as rainbow trout and scup. Spectral, electrophoretic, 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.