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G. Heo, N. Mast, W.-L. Liao, I. V. Turko, I. A. Pikuleva; Quantitative Analysis of Cholesterol-metabolizing Cytochrome P450 Enzymes in the Bovine Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2338.
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Cholesterol-metabolizing cytochrome P450 enzymes (CYPs 7A1, 11A1, 27A1, and 46A1) generate more polar forms of cholesterol for elimination or regulation. They convert cholesterol to 7-hydroxycholesterol (CYP7A1), pregnenolone (CYP11A1), 27-hydroxycholesterol (CYP27A1), and 24(S)-hydroxycholesterol (CYP46A1). Three cholesterol-metabolizing P450s (CYPs 27A1, 11A1, and 46A1) were reported to be expressed in the retina as assessed by immunocytochemical analysis. The expression levels of these enzymes in the retina, however, have not yet been assessed. In this study, we evaluated the expression levels of CYPs 11A1, 27A1, and 46A1 in the bovine retina and retinal pigment epithelium (RPE) by western blotting and mass spectrometry.
We purified recombinant CYPs and used them as standards for generation of the calibration curves. The IRDye goat anti-rabbit secondary antibody and Odyssey infrared imaging system (Li-COR biosciences) were used for quantitative western blotting. To quantify P450s by mass spectrometry, we used multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer and 15N-labeled recombinant CYPs as internal standards.
The results of the western blotting indicate that expression levels of CYPs in the bovine retina and RPE are low and less than 0.1 pmol (CYP11A1), 0.05 pmol (CYP27A1), and 0.01 pmol (CYP46A1) of P450 per 50 ug of protein. For comparison, the expression of CYP11A1 in the bovine adrenal glands and that of CYP27A1 in the bovine liver is 0.8 pmol and 0.18 pmol per 50 ug of mitochondrial protein, respectively. In mass spectrometry measurements, we determined the optimum pairs of precursor and fragment ions for the selected tryptic peptides from purified CYPs. Obtaining the absolute values of P450s with greater statistical power is in progress.
Expression levels of cholesterol-metabolizing P450s in the bovine retina cannot be reliably quantified by western blotting. More sophisticated techniques are required to accomplish this goal.
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