Retinyl Esters Are the Substrate for Isomerohydrolase

Abstract

Abstract: : Purpose: Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified. Both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study was designed to determine the substrate of the isomerase enzyme or enzymatic complex. Methods: LRAT and isomerase activities were measured in bovine microsomes and mouse eyecup homogenates using all-trans [H³]-retinol as a substrate. The highly specific LRAT inhibitor 10-N-acetamidododecyl chloromethyl ketone (AcDCMK) was synthetized. AcDCMK and cellular retinol-binding protein I (CRBP) were used to inhibit LRAT activity. The retinoid reaction products were extracted and analyzed by HPLC. Results: The specific LRAT inhibitors AcDCMK and CRBP decreased the generation of both retinyl esters and 11-cis retinol from all-trans retinol. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [³H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol.The RPE65 knockout (Rpe65^-/-) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65^-/- mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Conclusion: Our data indicate that retinyl esters are the substrate of the isomerization reaction.

Keywords: protein structure/function • retinoids/retinoid binding proteins • retinal pigment epithelium