| 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 beenidentified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. Thisstudy is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovineRPE microsomes with all-trans [3H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition oflecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) orcellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinolfrom all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not withthe all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to theaddition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubationof all-trans [3H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectableproduction 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 miceplateau at 8 weeks-of-age, but Rpe65-/- mice continue to accumulate retinyl esters with age (e.g., at 36weeks, the levels are 20× that of WT). Our data indicate that the retinyl esters are the substrate of theisomerization reaction.
|
