Publications

• Purified RPE65 shows isomerohydrolase activity after reassociation with a phospholipid membrane.

  Nikolaeva O., Takahashi Y., Moiseyev G., Ma J.X.

  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 react ... >> More

  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 isomerohydrolase 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. << Less

  FEBS J. 276:3020-3030(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Identification of key residues determining isomerohydrolase activity of human RPE65.

  Takahashi Y., Moiseyev G., Ma J.X.

  RPE65 is the retinoid isomerohydrolase that converts all-trans-retinyl ester to 11-cis-retinol, a key reaction in the retinoid visual cycle. We have previously reported that cone-dominant chicken RPE65 (cRPE65) shares 90% sequence identity with human RPE65 (hRPE65) but exhibits substantially highe ... >> More

  RPE65 is the retinoid isomerohydrolase that converts all-trans-retinyl ester to 11-cis-retinol, a key reaction in the retinoid visual cycle. We have previously reported that cone-dominant chicken RPE65 (cRPE65) shares 90% sequence identity with human RPE65 (hRPE65) but exhibits substantially higher isomerohydrolase activity than that of bovine RPE65 or hRPE65. In this study, we sought to identify key residues responsible for the higher enzymatic activity of cRPE65. Based on the amino acid sequence comparison of mammalian and other lower vertebrates' RPE65, including cone-dominant chicken, 8 residues of hRPE65 were separately replaced by their counterparts of cRPE65 using site-directed mutagenesis. The enzymatic activities of cRPE65, hRPE65, and its mutants were measured by in vitro isomerohydrolase activity assay, and the retinoid products were analyzed by HPLC. Among the mutants analyzed, two single point mutants, N170K and K297G, and a double mutant, N170K/K297G, of hRPE65 exhibited significantly higher catalytic activity than WT hRPE65. Further, when an amino-terminal fragment (Met(1)-Arg(33)) of the N170K/K297G double mutant of hRPE65 was replaced with the corresponding cRPE65 fragment, the isomerohydrolase activity was further increased to a level similar to that of cRPE65. This finding contributes to the understanding of the structural basis for isomerohydrolase activity. This highly efficient human isomerohydrolase mutant can be used to improve the efficacy of RPE65 gene therapy for retinal degeneration caused by RPE65 mutations. << Less

  J. Biol. Chem. 289:26743-26751(2014) [PubMed] [EuropePMC]

• Hydrolysis of 11-cis- and all-trans-retinyl palmitate by homogenates of human retinal epithelial cells.

  Blaner W.S., Das S.R., Gouras P., Flood M.T.

  The retinal epithelium plays an important role in the storage and metabolism of retinoids in the eye. Studies were conducted to examine the enzymatic hydrolysis of retinyl esters by human retinal epithelial cells. Homogenates prepared from these cells were found to hydrolyze both the 11-cis- and a ... >> More

  The retinal epithelium plays an important role in the storage and metabolism of retinoids in the eye. Studies were conducted to examine the enzymatic hydrolysis of retinyl esters by human retinal epithelial cells. Homogenates prepared from these cells were found to hydrolyze both the 11-cis- and all-trans-isomers of retinyl palmitate. Retinyl ester hydrolysis was time-, protein-, and pH-dependent. The 11-cis isomer was hydrolyzed at a rate which was approximately 20 times greater than that of the all-trans isomer. The 11-cis-retinyl palmitate hydrolase activity did not require detergents, unlike the all-trans-retinyl palmitate hydrolase activity, which required detergents for activity. The 11-cis-retinyl palmitate hydrolase activity was maximally active with the addition of 1.0% sodium taurocholate at about pH 8.5, was abolished by incubation at 50 degrees C for 10 min, and was quantitatively recovered in the pellet after centrifugation at 100,000 X g for 1 h. The rate of hydrolysis of 11-cis-retinyl palmitate became saturated with increasing concentrations of 11-cis-retinyl palmitate; under the assay conditions employed, the hydrolase activity had an apparent Km of 19 microM toward 11-cis-retinyl palmitate. All-trans-retinol and 11-cis-retinyl did not affect the rate of hydrolysis of 11-cis-retinyl palmitate, and addition of all-trans-retinyl palmitate only weakly inhibited the 11-cis-retinyl palmitate hydrolytic activities. These data indicate that the human retinal epithelium possesses distinct activities for the hydrolysis of 11-cis- and all-trans-retinyl esters and raise the possibility that these activities may provide a means of distinguishing the stereoisomers of retinol in this tissue. << Less

  J Biol Chem 262:53-58(1987) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.