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J.-X. Ma, O. Nikolaeva, Y. Takahashi, G. Moiseyev; Purified RPE65 Shows Isomerohydrolase Activity After Re-Association With Liposome. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1212.
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© ARVO (1962-2015); The Authors (2016-present)
Generation of 11-cis retinol from all-trans retinyl ester in the retinal pigment epithelium is a critical step in the visual cycle and is essential for perception of light. Recent studies from cell culture model systems suggest that protein RPE65 is the retinoid isomerohydrolase catalyzing the reaction. However, this enzymatic activity has never been observed in purified RPE65 previously, and thus, it remains controversial whether RPE65 is an enzyme. The purpose of this study is to demonstrate the isomerohydrolase activity using the purified RPE65.
Recombinant chicken RPE65 with a His-tag was expressed in 293A-LRAT cells using the adenovirus vector. Their expression levels were analyzed by Western blot analysis. The protein was dissociated from the membrane with CHAPS and purified through Ni-NTA affinity chromatography. To develop a novel liposome-based assay for isomerohydrolase, liposomes containing all-trans retinyl ester were formed. The binding of RPE65 to the liposomes was analyzed by a liposome flotation assay using gradient ultracentrifugation. Isomerohydrolase activity of the purified RPE65 was determined using the in vitro enzymatic assay, and the generated 11-cis retinol quantified by HPLC.
CHAPS concentration was optimized to achieve the efficient solubilization and purification of RPE65 while preserving its enzymatic activity. Recombinant chicken RPE65 was purified to apparent homogeneity. The purified RPE65 showed an efficient binding to the all-trans retinyl palmitate-containing liposomes. Upon binding to liposomes, the purified RPE65 generated 11-cis retinol, demonstrating isomerohydrolase activity in the purified RPE65. However, the purified RPE65 did not show any detectable enzymatic activity in the absence of the liposomes, suggesting the membrane association is essential for the enzymatic activity of RPE65. The all-trans retinyl ester is required to be incorporated in the phospholipid membrane to serve as a substrate for isomerohydrolase. This assay system using purified RPE65 enabled us to measure kinetic parameters for the enzymatic reaction catalyzed by RPE65, Km=3.7 µM and kcat=1.45x10-4 s-1.
These results provide a conclusive proof that RPE65 is the isomerohydrolase of the visual cycle. The liposome-based isomerohydrolase assay using purified RPE65 is a useful assay for further studies of this important enzyme.
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