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D. Choudhary, I. Jansson, J. Schenkman, M. Sarfarazi; Comparative analysis of orthologous CYP1B1 endobiotic metabolism . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2084.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Orthologs of cytochrome P450 1B1 (CYP1B1) in human and mouse are highly conserved, with 81% sequence identity. An absence of functional CYP1B1 in humans causes an improper development of the trabecular meshwork in the eye. A similar defect is seen in Cyp1b1–null mice. The present study was initiated to determine the substrate/metabolite of CYP1B1 associated with the primary congenital glaucoma phenotype. Methods: Endobiotic substrates of CYPs are components of intermediary metabolism of lipids, certain vitamins and steroids. Representative samples of each of these groups were selected and their metabolism by the mouse and human orthologs, Cyp1b1 and CYP1B1, respectively, were compared. Assays used purified recombinant enzymes in reconstituted systems, and metabolites were extracted and measured by high performance liquid chromatography (HPLC), mass spectrometry and thin layer chromatography. Results: Both CYP1B1 and Cyp1b1 orthologs were able to oxidize vitamin A (all–trans–retinol, ROL) to all–trans–retinal (RAL) and RAL to all–trans–retinoic acid (RA), an active morphogen. Neither ortholog metabolized RA. Substrate dependence determinations on the mouse Cyp1b1 revealed Km values for ROL and RAL approximately 20–fold higher than for CYP1B1. Biologically active metabolites (hydroxyeicosatetraenoic acids, HETEs, and epoxyeicosatrienoic acids, EETs) are produced from arachidonic acid by CYPs. In addition to exerting direct biological activity, these are also capable of evoking transcriptional regulation. Comparison of the metabolism of arachidonic acid by CYP1B1 and Cyp1b1 showed the human ortholog to have a 3.5–fold higher Vmax. Cyp1b1 had a 17–fold higher Km value for arachidonate. Similar differences between the mouse and human ortholog were also seen in the metabolism of ß–estradiol. Conclusions: The concept of using a comparison of metabolic activities between developmentally active, orthologous forms of cytochrome P450 is a new approach with the potential to reveal the identity of the active signaling compound in eye development. Supported By National Eye Institute (EY–11095)
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