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G. P. Moiseyev, Y. Takahashi, M. Kishinevsky, A. Albeck, R. K. Crouch, J.-X. Ma; Intact Retinol Polyene Side Chain is Essential for Isomerization, but Not Binding, by RPE65 Isomerohydrolase. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3519.
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Isomerization and hydrolysis of all-trans retinyl esters to 11-cis retinol is a critical step in the visual cycle for the regeneration of 11-cis retinal and is catalyzed by the RPE65 isomerohydrolase. The purpose of this study is to investigate the specificity of RPE65 towards retinol analogs with substitutions along the retinol polyene side chain.
RPE65 was expressed in the HEK293 cell line stably expressing lecithin retinol acyl transferase (LRAT) 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 analogs. The retinoid products were analyzed by HPLC and identified by the elution times and UV-VIS spectra. Inhibition of isomerohydrolase activity measured with all-trans [3H]-retinol as a substrate by unlabelled all-trans-retinol was used as a measure of the binding of the analogs to RPE65.
Adenovirus mediated high levels of RPE65 expression in HEK293A cells. The lysate from the HEK293-LRAT cells expressing RPE65 converted significant amounts of all-trans retinol to 11-cis retinol. However, all-trans-9-desmethyl retinol, all-trans-9-CF3 retinol, all-trans-13-CF3 retinol and all-trans-12-CN-13,14-dihydro retinol were all completely resistant to isomerization. All these retinol analogs were efficiently converted to the corresponding all-trans retinyl esters by LRAT. The inhibition assay demonstrated that all four analogues efficiently bind to RPE65.
The intact polyene moiety of retinol analog is essential for the efficient isomerization of the 11 double bond by RPE65 but not for the binding to RPE65 or esterification by LRAT
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