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G. Heo, N. Mast, I. A. Pikuleva, W.-L. Liao, I. V. Turko; Effect of Unique Retinal Environment on Catalytic Properties of Cholesterol-Metabolizing P450s. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5951.
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Mitochondrial cholesterol-metabolizing P450s 27A1 and 11A1 convert cholesterol to 27-hydroxycholesterol and pregnenolone, respectively. These enzymes are expressed in the retina, yet very little is currently known whether unique composition of retinal phospholipids (PLs), rich in n-3 polyunsaturated fatty acids, and retina-specific P450/redox partner ratio affect catalytic efficiencies and product profiles of P450s 27A1 and 11A1.
Kinetic studies were carried out in the in vitro reconstituted system containing purified recombinant P450, PLs extracted from either neural retina or retinal pigment epithelium (RPE), and the P450 redox partners adrenodoxin reductase and adrenodoxin. Catalytic properties of P450s were compared with those in the presence of PLs extracted from the adrenal and liver mitochondria, the classical sites of expression of CYP11A1 and CYP27A1, respectively. Quantification of P450s and their redox partners was carried out by Western blot and mass spectrometry using multiple reaction monitoring.
The catalytic efficiency of CYP27A1 (the kcat to Km ratio) was 1.7-fold higher with retinal PLs than with liver PLs, and the catalytic efficiency of CYP11A1 was 1.4-fold lower with retinal PLs than with adrenal PLs. The ratios of the P450s and their redox partners in the neural retina, RPE, adrenals, and liver were then determined and used to measure the enzymes’ turnover numbers under the conditions that model those in the retina and RPE. The turnover number of CYP27A1 with retinal PLs and retina-specific P450/redox partner ratio was at least 3-fold higher than that with the liver PLs and redox partner ratio, and the turnover number of CYP11A1 with retinal PLs was similar to that with adrenal PLs.
The data obtained suggest that CYP27A1 has a potential to be the major P450 contributing to cholesterol elimination from the retina and RPE.
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