Identification of an Intracellular Retinyl Ester Binding Protein in Bovine Retinal Pigment Epithelium

Abstract

Abstract: : Purpose: Although retinyl esters (RE) exist in large quantity in the retinal pigment epithelium (RPE), little is known about the transfer of RE between cytosol and membrane compartments. The focus of the present study is to identify carrier proteins which bind RE in the RPE. Methods: RPE and choroid were removed from freshly dissected bovine eye cups and RPE cells were collected by rinsing with isotonic buffer. Cells were then homogenized, sonicated, and centrifuged to remove membranes. Three ml supernatant (7.4mg protein/ml) fractions were incubated with [₃H]- all-trans retinyl palmitate (ATRP), [₃H] 11-cis retinyl palmitate (11-cis-RP), or ₁₄C palmitic acid (PA) at 4° C for 45 minutes. A separate fraction was also incubated with both (labeled) PA and ATRP (or 11-cis RP). After incubation, cytosolic proteins were applied to a 65 cm x 1.5 cm gel filtration column (S200), and then eluted with 0.25 M Tris-HOAc, 1 mM DTT, 2 mM EDTA, at a flow rate of 1.0 ml/min, pH 7.4. These experiments were repeated with RPE collected from eyes pre-washed with isotonic buffer to remove extracellular proteins. Eluted fractions were monitored for radioactivity by scintillation counting and for proteins with the Bradford method. Results: Column profiles obtained from cytosolic proteins incubated with either RE had a protein band with high molecular weight (eluting at void volume) co-eluting with the [₃H] labels of 11-cis or all-trans RP. Another protein band with a much lower molecular weight was also observed and it coeluted with the ₁₄C label of PA. Cytosolic proteins incubated with both (labeled) RP and PA similarly produced two distinct protein bands associating with the respective labels of RE and PA. The amount of radiolabels (in total DPM) associated with both protein bands did not change in the column profiles from RPE of pre-washed eyecups. Conclusion: Bovine RPE has separate intracellular binding proteins for PA and RP. The binding protein for PA has a low molecular weight. A novel binding protein of a higher molecular weight for RP is identified in the present study. Further characterization of this binding protein will be carried out to study its role in the visual cycle.

Keywords: 526 protein purification and characterization • 527 protein structure/function • 567 retinal pigment epithelium