Analysis of the Retinoid Isomerase Activities in the Retinal Pigment Epithelium and Retina

Light sensitivity in the vertebrate retina is mediated by the opsin visual pigments inside rod and cone photoreceptor cells. These pigments consist of a G protein-coupled receptor and the photo-sensitive ligand, 11-cis -retinaldehyde (11-cis- RAL). Absorption of a photon by an opsin pigment induces isomerization of the 11-cis- RAL chromophore to all-trans- retinaldehyde (all-trans- RAL), rendering the pigment insensitive to light. The bleached opsin regains light sensitivity by recombining with another 11-cis- RAL. The vertebrate eye contains a biochemical mechanism for regenerating 11-cis- RAL chromophore from all-trans- RAL, called the visual cycle. The visual cycle takes place within cells of the retinal pigment epithelium (RPE). A second visual cycle also appears to be present in M�ller glial cells of the retina. A critical step in the regeneration of 11-cis- RAL chromophore is thermal re-isomerization to the 11-cis configuration of an all-trans- retinyl ester (all-trans- RE) or an all-trans- retinol (all-trans- ROL). In RPE cells, this step is carried out by an enzyme called Rpe65 isomerase. This chapter provides methods for assaying Rpe65 isomerase. Although Rpe65 utilizes an all-trans- RE such as all-trans- retinyl palmitate (all-trans- RP) as substrate, it can be assayed in RPE homogenates by providing all-trans- ROL substrate and allowing the endogenous lecithin:retinol acyl transferase (LRAT) to synthesize all-trans- REs using fatty acids from phosphatidylcholine in the membranes. Alternatively, all-trans- RP can be provided directly as substrate, although this requires the isomerase reaction to be carried out in the presence of detergent, since fatty-acyl esters of all-trans- ROL are insoluble. Methods are provided in this chapter for assaying Rpe65 in RPE homogenates with both all-trans- ROL and all-trans- RP substrates. A second visual cycle appears to be present in the retinas of cone-dominant species such as chicken. This retinal pathway may augment the RPE to provide 11-cis- RAL to cone photoreceptors under conditions of bright light where the rate of opsin photoisomerization is high. The isomerase in this pathway (isomerase-2) utilizes all-trans- ROL and palmitoyl coenzyme A (palm CoA) as substrates to synthesize 11-cis- retinyl palmitate (11-cis- RP). Isomerase-2 appears to be present in M�ller cells but has not yet been identified. Methods are provided in this chapter for assaying isomerase-2 in chicken retina homogenates.