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G. P. Moiseyev, Y. Takahashi, Y. Chen, J.-X. Ma; RPE65 From Cone-Dominant Chicken Has a Higher Isomerohydrolase Activity Than Bovine and Human RPE65. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3251.
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Cones recover their sensitivity significantly faster than rods after bleaching. It has been suggested that a higher-rate regeneration of 11-cis retinal chromophore is required for cones to continuously function under bright light conditions. We proposed that the isomerohydrolase RPE65, an enzyme catalyzing a key step in regeneration of 11-cis retinal, is more efficient in the RPE of cone-dominant chicken than rod-dominant species. The purpose of this study was to compare the specific isomerohydrolase activity of RPE65 from chicken RPE with that from rod-dominant bovine and human.
The human and chicken RPE65 cDNAs were cloned to construct adenoviruses expressing these RPE65 under a CMV promoter. Proteins were expressed in 293-LRAT cells, a stable cell line expressing human LRAT, and expression levels of recombinant RPE65 were quantified by Western blot analyses using purified chicken and bovine RPE65 as protein standards for the quantification. The enzymatic activities were measured by in vitro activity assays using microsomal proteins and analyzed by HPLC. The isomerohydrolase activity was normalized by amount of RPE65 in the assay.
The abundance of RPE65 in the chicken RPE microsomes was 5-fold lower than that in bovine RPE microsomes. Similar to that of human and bovine RPE65, the isomerohydrolase activity in chicken RPE was blocked by specific LRAT inhibitors AcDCMK and apo-CRBP, suggesting chicken RPE65 also uses all-trans retinyl ester as its direct substrate. The rate of generation of 11-cis retinol was measured in bovine and chicken RPE microsomes. After normalization of the initial retinol isomerization rate by the RPE65 content in RPE microsomes, specific enzymatic activity of native chicken RPE65 was 11.7-fold higher than that of bovine RPE65. Adenoviral vector mediated a high level expression of chicken and human RPE65 in 293-LRAT cells. Isomerohydrolase activity of recombinant chicken RPE65 was 7.7-fold higher than that of recombinant human RPE65 under the same conditions, after normalization by RPE65 levels.
Chicken RPE65 possesses significantly higher specific retinol isomerohydrolase activity, when compared to bovine and human RPE65, suggesting that cone-dominant chicken has a faster retinoid cycle than rod-dominant species. This finding correlates well with the faster regeneration rates of visual pigments in cone-dominant retinas.
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