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. 2010:652:329-39.
doi: 10.1007/978-1-60327-325-1_19.

Analysis of the retinoid isomerase activities in the retinal pigment epithelium and retina

Gabriel H Travis  1 , Joanna Kaylor, Quan Yuan
Affiliations

Analysis of the retinoid isomerase activities in the retinal pigment epithelium and retina

Gabriel H Travis et al. Methods Mol Biol. 2010.

Abstract

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 Müller 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 Müller cells but has not yet been identified. Methods are provided in this chapter for assaying isomerase-2 in chicken retina homogenates.

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Figures

Fig. 19.1
Fig. 19.1
Visual cycle in RPE cells. 11-cis-RAL in rhodopsin is isomerized by absorption of a photon (hv). The resulting all-trans-RAL is reduced to all-trans-ROL by all-trans-RDH in the rod outer segment. The all-trans-ROL is taken up by an RPE cell where it is esterified to a fatty acid by LRAT. The resulting all-trans-RE is isomerized and hydrolyzed by Rpe65 to 11-cis-ROL. The 11-cis-ROL is oxidized to 11-cis-RAL by 11-cis-RDH. Both 11-cis-ROL and 11-cis-RAL are bound to CRALBP within the RPE cell. During transit through the extracellular space, all-trans-ROL and 11-cis-RAL are bound to IRBP (not shown).
Fig. 19.2
Fig. 19.2
Representative retinoid chromatograms. (a) Chromatogram of retinoid standards showing UV absorption at 318 nm. Identified 11-cis-RP, all-trans-RP, 11-cis-RAL, all-trans-RAL, 11-cis-ROL, and all-trans-ROL peaks are labeled. (b) Chromatogram of chicken retina homogenate in the isomerase-2 assay mixture before incubation (t 0) showing 20–25 min elution times. This chromatogram shows endogenous 11-cis-ROL and 13-cis-ROL, plus all-trans-ROL substrate (off scale) added to the assay mixture. (c) Chromatogram of chicken retina homogenate in isomerase-2 assay mixture following 15 min incubation at 37°C (t15–min). Note the higher 11-cis-ROL peak. The difference in 11-cis-ROL before and after incubation reflects the isomerase-2 activity. 13-cis-ROL increases during the incubation due to thermal isomerization of 11-cis-ROL product and all-trans-ROL substrate. (d) UV spectrum of 11-cis-ROL. The wavelength of maximum absorption (λmax) for 11-cis-ROL is 318 nm. (e) UV spectrum of 13-cis-ROL (λmax, 328 nm). (f) UV spectrum of all-trans-ROL (λmax, 325 nm).
Fig. 19.3
Fig. 19.3
Rpe65 and isomerase-2 activities in chicken retina and RPE. (a) Isomerase-specific activities (pmol 11-cis-ROL per min per mg protein) in chicken retina and chicken RPE homogenates using the Rpe65 assay with all-trans-RP substrate. Rpe65 activity is almost undetectable in retina homogenates. (b) Isomerase-specific activities in chicken retina and chicken RPE homogenates using the isomerase-2 assay with all-trans-ROL and palm CoA substrates. Retinoids were saponified after assay incubation. Note the much higher activity in chicken retina versus RPE homogenates. Also note the difference in scale of specific activities between figure panels (a) and (b). During dissection, all visible traces of pigmented RPE material were removed from the retina samples. However, contaminating retina could not be removed from the RPE samples since retina is non-pigmented. Thus, RPE samples in this experiment probably contained contaminating retina material, while the retina samples were relatively free of RPE contamination.
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