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A. Muniz, A. Mukherjee, A. Hatch, E. T. Villazana-Espinoza, D. Allen, A. T. C. Tsin; Isomerization of All-Trans Retinol to 11-cis Retinol in the Cone Dominant Mammalian Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3250.
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Isomerization of all-trans retinol to 11-cis retinol in the mammalian cone dominant retina has previously been reported. However, this isomerase activity has not been characterized. The purpose of this study is to characterize this isomerase activity in the cone dominant mammalian retina.
Retina and RPE from 13 lined ground squirrel were freshly collected and whole membranes were prepared. Retinal membrane proteins at a concentration of 600µg/ml was incubated with 30µM CRALBP, 50µM all-trans retinol, and 1% BSA in reaction buffer at 37ºC for 5, 10, 20, 30 and 40 min. RPE membranes were similarly incubated with substrate and other factors for 30 min. The final reaction volume was 0.5 ml. The isomerization reaction was stopped with ice cold ethanol, and retinol was extracted with hexane, and analyzed by HPLC. Retinols were identified by retention time against authentic standards and by absorption spectra. We also analyzed retinal and RPE protein samples from the ground squirrel for RPE65 by Western blot.
From the time course plot, we determined that 30 minutes was optimal incubation for retinal membrane isomerase activity (n=4). The activity yield at this incubation time is 18 pmol/mg/min. We then tested the RPE membrane preparation under the same conditions and the isomerase activity in the RPE was 12 pmol/mg/min (n=2). The Western blot results show the presence of RPE65 in both the retina and RPE of the ground squirrel.
The cone visual cycle in the cone dominant mammalian retina requires the support of an active isomerase which has not be characterized. Results from the present study support the suggestion that a retinol isomerase may exist in the cone dominant ground squirrel retina. Furthermore, we also found a similar but somewhat lower isomerase activity in the adjacent RPE, suggesting that these tissues may work together to provide 11-cis retinoid for cone pigment regeneration in the retina. Immunochemical studies showed the presence of RPE65 in both the ground squirrel retina and RPE suggesting that RPE65 may be responsible for this activity.
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