Chicken Retinas Contain a Retinoid Isomerase Activity That Catalyzes the Direct Conversion of All–Trans–Retinol to 11–Cis–Retinol

Abstract

Abstract: : Purpose: Regeneration of 11–cis–retinaldehyde (11cRAL) chromophore following light absorption involves an enzyme pathway called the visual cycle in the RPE. The isomerase in this pathway uses all–trans–retinyl esters (atRE’s) synthesized by lecithin:retinol acyl transferase (LRAT) as substrate. Several lines of evidence suggest that cone opsins may regenerate by a different mechanism. Methods: We separated retinas from the RPE of dissected chicken and bovine eyes and prepared microsomal membranes for biochemical analysis. We incubated chicken retina and bovine RPE membranes with all–trans–retinol (atROL) or 11–cis–retinol (11cROL) substrates and analyzed for production of new retinoids by HPLC. Results: Bovine RPE membranes synthesized predominantly atRE’s from atROL substrate. In contrast, chicken retina membranes synthesized significantly more 11–cis–retinyl esters (11cRE’s). Synthesis of 11cRE’s preceded appearance of atRE’s. Immunoblot analysis of chicken and bovine retina and RPE samples showed that LRAT is expressed in RPE from both species, but is undetectable in retinas. Addition of atROL to media containing chicken retina explants resulted in synthesis of 11cRE’s followed by 11cRAL. Levels of atRE’s were negligible throughout the incubation. Acyl CoA:retinol acyltransferase (ARAT) is another retinyl ester synthase that uses palmitoyl coenzyme A (palm CoA) as an acyl donor and is inhibited by progesterone. We incubated chicken retina membranes with atROL plus or minus palm CoA, cellular retinaldehyde binding protein (CRALBP), and progesterone, and assayed for formation of new retinoids. Palm CoA strongly stimulated synthesis of atRE’s and 11cRE’s. Addition of apo–CRALBP to the incubation mixture stimulated synthesis of 11cROL four–fold. We observed 18–fold suppression of atRE–synthesis and two–fold suppression of 11cRE–synthesis with the addition of progesterone. Importantly, formation of 11cROL increased three–fold in the presence of progesterone. Conclusions: (1) Cone–dominant chicken retinas contain a retinoid isomerase activity distinct from the isomerase in RPE. This isomerase in chicken retinas catalyzes the direct conversion of atROL to 11cROL. (2) Chicken retinas contain ARAT activity but not LRAT. (3) The formation of 11–cis–retinoids from atROL by chicken retina membranes is driven by mass action through secondary esterification of the 11cROL product. (4) We suggest that the atROL isomerase and ARAT are components of an alternate visual cycle that mediates opsin regeneration in cones.

Keywords: retinoids/retinoid binding proteins • color pigments and opsins • retinal pigment epithelium