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J.J. Kaylor, M. Jin, W. Moghrabi, G.H. Travis; Strategies for Cloning the Retinol Isomerase in Cone–Dominant Chicken Retinas . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3740.
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© ARVO (1962-2015); The Authors (2016-present)
The visual cycle in RPE cells for the regeneration of rhodopsin is well defined. However, several lines of evidence suggest that cones may regenerate visual pigments by a mechanism independent of the RPE. Prior work from our laboratory indicated the existence of a retinoid isomerase (distinct from Rpe65) in chicken and ground squirrel retinas that catalyzes the direct conversion of all–trans–retinol (atROL) to 11–cis–retinol (11cROL). This activity is coupled to a palmitoyl coenzyme A (palm CoA) dependent retinyl ester synthase to yield 11–cis–retinyl palmitate (11cRP) as a final product. We are currently working to clone this retinol isomerase and retinyl ester synthase.
We prepared homogenates, total membranes, and microsomes from chicken retinas, bovine retinas, and bovine RPE. These samples were used as enzyme sources in assay mixtures containing atROL and palm CoA. Following incubation, the reactions were quenched with methanol, extracted with hexane, and analyzed for the production of 11–cis–retinoids by HPLC. A normalized cDNA library from chicken retinas was prepared in a mammalian expression plasmid. We also prepared a cell line (293T–C) that stably expresses cellular retinaldehyde binding protein (CRALBP). We have begun screening pools of clones from the chicken retina library transfected into 293T–C cells. After incubating in medium containing atROL, we isolate the cells, homogenize, extract with hexane, and analyze by HPLC.
Homogenates and membrane fractions from chicken retinas catalyzed the synthesis of 11cROL and 11–cis–retinyl palmitate (11cRP) from added atROL and palm CoA. We have identified a palm CoA–dependent retinyl ester synthase activity in 293T–C cells transfected with pools of the chicken library. Library screening is ongoing to identify an isomerase activity in these transfected cells. We are also exploring an in vitro approach to screen the library for the retinol isomerase.
Library screening has revealed the presence of a palm CoA–dependent retinyl ester synthase in chicken retinas. This may be identical to the acyl CoA:retinol acyltransferase (ARAT) activity described in multiple tissues including RPE.
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