tailieunhanh - Báo cáo khoa học: Purified RPE65 shows isomerohydrolase activity after reassociation with a phospholipid membrane

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 findings from cell culture models suggest that protein RPE65 is the retinoid isomerohydrolase that catalyzes the reaction. However, previous attempts to detect the enzymatic activity of purified RPE65 were unsuccessful, and thus its enzymatic function remains contro-versial. | ễFEBS Journal Purified RPE65 shows isomerohydrolase activity after reassociation with a phospholipid membrane Olga Nikolaeva Yusuke Takahashi Gennadiy Moiseyev and Jian-xing Ma Departments of CellBiology and Medicine Endocrinology Harold Hamm Oklahoma Diabetes Center University of Oklahoma Health Sciences Center OK USA Keywords isomerohydrolase liposome retina retinyl ester RPE65 Correspondence G. Moiseyev Departments of CellBiology and Medicine Endocrinology Harold Hamm Oklahoma Diabetes Center University of Oklahoma Health Sciences Center 941 Stanton L. Young blvd BSEB 302 Oklahoma City Ok 73104 USA Fax 1 405 271 3973 Tel 1 405 2718001 ext. 48443 E-mail gennadiy-moiseyev@ Received 3 February 2009 revised 23 March 2009 accepted 24 March 2009 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 findings from cell culture models suggest that protein RPE65 is the retinoid isomerohydrolase that catalyzes the reaction. However previous attempts to detect the enzymatic activity of purified RPE65 were unsuccessful and thus its enzymatic function remains controversial. Here we developed a novel liposome-based assay for isomerohy-drolase activity. The results showed that purified recombinant chicken RPE65 had a high affinity for all-trans-retinyl palmitate-containing liposomes and demonstrated a robust isomerohydrolase activity. Furthermore we found that all-trans-retinyl ester must be incorporated into 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. These results provide conclusive evidence that RPE65 is the isomerohydrolase of the visual cycle. doi In vertebrates both rod and cone visual pigments require 11-cis-retinal as a chromophore 1 . Upon absorption