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J.–X. Ma, Y. Chen, Y. Takahashi, G. Moiseyev; RPE65 Is the Isomerohydrolase in the Retinoid Visual Cycle . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1057.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Isomerization of all–trans retinoids to 11–cis isoforms is a critical step in the visual cycle and is catalyzed by a yet to be identified enzyme, isomerohydrolase. RPE65 is predominantly expressed in the RPE and is essential for the isomerization step. The purpose of this study is to investigate the role of RPE65 in the isomerization process. Methods: RPE65 and retinol acyltransferase (LRAT) were co–expressed in HEK293 cells using adenovirus and plasmid vectors. The expression of both the proteins was confirmed by Western blot analysis. The subcellular localization was revealed by immunocytochemistry using anti–RPE65 and anti–LRAT antibodies, respectively. The isomerohydrolase activity was measured in cell lysates using all–trans [3H] retinol as a substrate. The retinoid products were analyzed by HPLC with a flow scintillation analyzer. Results: Adenovirus mediated high levels of RPE65 expression in HEK293 cells. RPE65 and LRAT showed similar subcellular localization. The lysate from cells expressing both RPE65 and LRAT converted significant amounts of all–trans retinol to 11–cis retinol, demonstrating robust isomerohydrolase activity. The isomerohydrolase activity correlated with the expression levels of RPE65. In contrast, the cells infected with the control virus (GFP) did not show any activity. Moreover, expression of only RPE65 without LRAT did not show any isomerohydrolase activity under the same conditions. Addition of cellular retinaldehyde–binding protein (CRALBP) to the reaction system is not critical for the isomerohydrolase activity, although it increased isomerohydrolase activity. Conclusions: RPE65 is the long sought isomerohydrolase in the visual cycle. It requires the presence of LRAT in the same location to generate retinyl ester in situ as substrate for RPE65.
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