Cytochrome P4502B-type protein from feral leaping mullet (Liza Saliens) liver microsomes: its isolation and biochemical and immunological characterization

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2002
Bozcaarmutlu, Azra
Cytochrome P450s constitute a superfamily of hemoproteins that play important roles in oxidative metabolism of endogenous and exogenous compounds, and serve as catalysts that are significant in numerous and diverse biological pathways. The extent to which these various pathways or functions occur in different animal groups depend on a large degree on the participation of different P450 proteins present, their catalytic function and their regulation. Cytochrome P4502B subfamily is generally associated with detoxifying metabolism of xenobiotics in experimental animal species and also metabolizes hormones, testosterone and androstedione and several important pharmaceutical agents. The important role of cytochrome P4502B subfamily has been recognized by many biochemists, pharmacologists and toxicologist and has been studied in many different mammalian species. There could be a large number of homologous genes in fish. inIn this study, basic cytochrome P450 that cross-reacted with antibody produced against cytochrome P4502B4 homologue, eluted from the first DEAE- cellulose column was purified to electrophoretic homogeneity by using first and second anion exchange chromatography, and three successive hydroxylapatite column chromatographies. Purified cytochrome P450 was characterized with respect to spectral, electrophoretic, immunochemical and biocatalytic properties. Purified cytochrome P450 gave a single band on SDS-polyacrylamide gel electrophoresis having monomer molecular weight of 49300 Da. Absolute absorption spectrum of the purified cytochrome P450 showed maximum at 417 ran and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. Purified cytochrome P450 was found to be active in the N- demethylation of benzphetamine, cocaine, erythromycin and ethylmorphine and O-depenthylation of pentoxyresorufin in the reconstituted systems containing purified cytochrome P450 reductase from leaping mullet and synthetic lipid. However, it was unable to catalyze the oxidation of the other monoxygenase substrates such as aniline, lauric acid, 7-ethoxyresorufin, 7-methoxyresorufin and 7-benzyloxyresorufin. Benzphetamine N-demethylase activity was inhibited with proadifen, a cytochrome P4502B inhibitor in reconstituted system. In addition, purified cytochrome P450 showed strong cross-reactivity with the antibody produced against sheep lung cytochrome P4502B4 homologue. The results obtained after accomplishing above studies, strongly suggested that cytochrome P450 purified from leaping mullet is a cytochrome P4502B-type protein. In this study, for the first time, a member of cytochrome P4502B subfamily was purified and characterized in fish species.

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Citation Formats
A. Bozcaarmutlu, “Cytochrome P4502B-type protein from feral leaping mullet (Liza Saliens) liver microsomes: its isolation and biochemical and immunological characterization,” Ph.D. - Doctoral Program, Middle East Technical University, 2002.