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G.P. Moiseyev, Y. Chen, R.K. Crouch, J.–X. Ma; Iron Is Required for the Isomerohydrolase Activity in the Visual Cycle . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1056.
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Purpose: The isomerization of all–trans retinol to 11–cis retinol in the RPE is a critical step in the visual cycle and is essential for normal vision. The isomerohydrolase which catalyzes this reaction has not been identified, despite intensive efforts, and many of its features remain unknown. This study aims to investigate if metal ions are required for the isomerohydrolase activity. Methods: All–trans [3H] retinol was used as a substrate to measure the activities of lecithin:retinol acyltransferase (LRAT) and isomerohydrolase in bovine RPE microsomes. Endogenous metal ions in the microsomes were deprived using metal chelators EDTA and 1,10–phenantroline. The retinoids generated were extracted and analyzed by HPLC with a flow scintillation analyzer. Results: Both the chelators showed dose–dependent inhibitions of the isomerohydrolase activity, with an IC50 of 125 mM for EDTA and 0.2 mM for 1,10–phenantroline. In the same reaction system, LRAT activity was not affected by either of these metal chelators. The retinol isomerase activity inhibited by the metal chelators was restored by the addition of FeSO4 but not by CuSO4, ZnCl2 or MgCl2. Moreover, addition of Fe(III) citrate or FeCl3 did not restore the isomerase activity, suggesting that ferrous ion is essential for the isomerohydrolase activity. Deprivation of iron from mice by an iron–deficient diet resulted in a significant decrease of isomerohydrolase activity in the mouse RPE. To determine if the Fe2+ is bound to RPE65 protein that is essential for the isomerization step, we stained purified bovine RPE65 for iron in gel using two specific iron–staining methods. Both methods demonstrated that RPE65 contains endogenous iron. Conclusions:This study indicates that endogenous Fe2+ is essential for the isomerohydrolase activity and RPE65 is an iron–containing protein.
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