Retinyl Esters Are the Substrate for Isomerohydrolase

Abstract

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 and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. 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 [(3)H]-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). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.

RPE65 from Cone-dominant Chicken Is a More Efficient Isomerohydrolase Compared with That from Rod-dominant Species

“…2). The identity of the purified RPE65 was confirmed by Western blot analysis as described previously (16).…”

“…We measured the dependence of chicken isomerohydrolase activity upon the generation of alltrans-retinyl ester using two specific LRAT inhibitors, apo-cellular retinol-binding protein Type 1 (apo-CRBP) and 10-N-acetamidododecyl chloromethyl ketone (Ac-DCMK), which have been characterized previously (16,21). In the absence of the inhibitors, a significant amount of 11-cis-retinol was generated from all-trans-retinol after 1.5-h incubation with bovine or chicken RPE microsomes (Fig.…”

“…Recombinant chicken RPE65 was purified from 293A cells infected by the adenovirus-expressing chicken RPE65 (Ad-chRPE65) at multiplicity of infection of 100 for 24 h. Microsomes were prepared from the infected 293A cells as described previously (16). The microsomal proteins were solubilized in 1% CHAPS and centrifuged at 100,000 ϫ g for 1 h. The supernatant was applied onto a DEAE-Sepharose column (1 ϫ 20 cm).…”

“…Un-infected 293A-LRAT cells were used as a negative control. Cell lysates were sonicated on ice for 20 s in a reaction buffer (10 mM BTP, pH 8.0, 100 mM NaCl), and microsomal fractions were prepared as described previously (16). Briefly, sucrose was added to the cell homogenate to the concentration of 0.32 M. The homogenate was centrifuged (20 min, 20,000 ϫ g) to sediment unbroken cells, nuclei, and mitochondria.…”

“…All-trans- [11,12- l of cold methanol and 300 l of hexane and centrifuged at 10,000 ϫ g for 5 min. The generated retinoids were analyzed by normal phase HPLC as described (16). The peak of each retinoid isomer was identified based on the retention time of retinoid standards.…”