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W. Jahng, N. Fishkin, H. Matsuda, N. Nesnas, K. Nakanishi, R.R. Rando; Establishing a Proteomic Inventory of 11–cis–Retinoid Binding Proteins in RPE . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2968.
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© ARVO (1962-2015); The Authors (2016-present)
In order to identify 11–cis–retinoid binding proteins in RPE membranes, an affinity labeling approach is used here which previously proved successful in functional identification of other critical visual cycle components, including lecithin retinol acyltransferase (LRAT) and RPE65. In these experiments, a specific alkylating affinity labeling reagent is used to label the target proteins in RPE and to subsequently identify them. In the present studies, a biotinylated 11–cis 7 membered ring containing retinoid (11cBRBA) is used to inactivate isomerase activity in RPE membranes. We have already shown that 11–cis–retinyl bromoacetate (11cRBA) is an irreversible inactivator of isomerization in functional assays using RPE membranes. The targets for this probe should help to identify those components necessary for the key trans to11–cis isomerization of vitamin A needed for continued vision. The labeling studies identify several of the visual cycle proteins already known to be important for isomerization, including CRALBP, LRAT, 11–cis–retinol dehydrogenase, RPE65, RGR, and GSTs.
RPE membranes were incubated with 11cBRBA (10 µM) for an hour at 4 oC to label the specific 11–cis–retinoid binding proteins. The synthesized ester bond containing retinoid was removed under basic conditions after labeling. Inhibition kinetic study of isomerohydrolase activity was also performed using 2 µM of 11cBRBA. Characterization of the labeled proteins were performed by avidin affinity purification, SDS–PAGE, Western blot, biotin blot and mass spectrometry analysis.
An integrated approach to classify 11–cis–retinoid binding proteins in RPE was developed by using a novel biotinylated retinoid with a constrained 11–cis geometry. This compound also showed strong inhibition (75%) of 11–cis–retinol biosynthesis in RPE in vitro. This method reveals that RPE65, 11cRDH, RGR, LRAT, GSTM, and GSTP binds 11–cis–retinoid specifically.
11cBRBA appears to be the first light–stable, irreversible inhibitor of 11–cis–retinol biosynthesis in RPE. The cycloheptenyl ring of this compound effectively locks the retinoid in the 11–cis geometry allowing it be recognized by the proteins involved in the retinoid cycle. Grant Identification: NIH Grant EY04096
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