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G.P. Moiseyev, Y. Takahashi, R.K. Crouch, J.–X. Ma; Isomerization of Retinol Analogs by Recombinant RPE65 Expressed in 293 Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2042.
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© ARVO (1962-2015); The Authors (2016-present)
Isomerization of all–trans retinol to 11–cis retinol is a key reaction in the visual cycle and is catalyzed by RPE65, an iron–dependent isomerohydrolase. The mechanism of the isomerization reaction is unknown. The purpose of this study is to investigate the effect of C20 methyl group removal on the isomerization of this retinol analog by recombinant RPE65.
RPE65 was expressed in 293 cell line stably expressing LRAT (lecithin retinol acyl transferase) using an adenovirus vector. The expression of both proteins was confirmed by Western blot analysis. The isomerohydrolase activity was measured in cell lysates using all–trans retinol and its analog. The retinoid products were analyzed by HPLC and identified by the elution times and UV–VIS spectra.
Adenovirus mediated high levels of RPE65 expression in 293A cells. The lysate from cells expressing both RPE65 and LRAT converted significant amounts of all–trans retinol to 11–cis retinol. All–trans–13–desmethyl retinol (lacking the C20 methyl group) was isomerized to its corresponding 11–cis–alcohol at a significantly lower rate (approximately 15–fold slower than unmodified all–trans–retinol). In contrast, both retinol analogs analyzed were efficiently converted to the corresponding all–trans retinyl esters by LRAT expressed in 293 cells.
The intact polyene moiety of retinol analog is essential for the efficient isomerization by RPE65.
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