Chemical Labeling of RPE Proteome by 11–cis–Retinyl Bromoacetate: Toward Characterization of the Putative Retinoid Isomerase

Abstract

Abstract: : Purpose: Isomerohydrolase activity is irreversibly inhibited in a saturable fashion by the affinity labeling agent 11–cis–retinyl bromoacetate (cRBA). Proteins of the retinal pigment epithelium (RPE) labeled by [³H]–cRBA are expected to have an essential role to play in 11–cis–retinol biosynthesis in the RPE. To begin to characterize isomerohydrolase in the RPE, specific affinity labeling experiments are carried out by incubating crude RPE membranes with [³H]–cRBA and the labeled proteins are identified. Methods: In this study, crude bovine RPE proteome was labeled by [³H]–cRBA. One and two–dimensional SDS–PAGE, trypsin digestion, followed by mass spectrometric analysis (ESI–MS/MS) reveal several 11–cis–retinoid specific binding proteins. Results:Cellular retinaldehyde binding protein (CRALBP), 11–cis–retinol dehydrogenase, RPE G–protein coupled receptor (RGR) and a few hypothetical proteins were specifically labeled by low concentrations (1 uM) [³H]–cRBA. The possiblity that some of the labeled proteins are isomerohydrolase candidates is being evaluated. Conclusions: Incubation of crude RPE proteins with [³H]–cRBA reveals the presence of several 11–cis–retinoid binding proteins which may be isomerohydrolase candidates.

Keywords: retinal pigment epithelium • retinoids/retinoid binding proteins • enzymes/enzyme inhibitors