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chunhe chen, Yiannis Koutalos, Molly Sprada, Federico Gonzalez-Fernandez; Removal Of All-Trans Retinol From Isolated Mouse Rod Photoreceptors By Interphotoreceptor Retinoid-Binding Protein (IRBP). Invest. Ophthalmol. Vis. Sci. 2012;53(14):3347.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the removal of the all-trans retinol formed after rhodopsin bleaching in the outer segment of single living mouse rod photoreceptors. All-trans retinol is formed in outer segments from the reduction of the all-trans retinal released from photoactivated rhodopsin; it is removed from outer segments by IRBP, a specialized carrier present in the interphotoreceptor matrix (IPM).
Native IRBP was extracted from the soluble IPM fraction of bovine retinas, and purified by a combination of concanavalin-A affinity, ion exchange, and S-300 size-exclusion chromatography. The concentration of the purified IRBP was determined by absorbance spectroscopy, and amino acid analysis. Experiments were carried out with dark-adapted living rod photoreceptors isolated from 2-3 months old 129/sv wild type mice. The amount of all-trans retinol in the outer segment was measured from its fluorescence (excitation 360 nm; emission >420 nm). Different concentrations of IRBP were added to the extracellular solution 30 min after bleaching and the removal of all-trans retinol was monitored from the decline in outer segment fluorescence. In some experiments IRBP was present in the extracellular solution prior to bleaching. Experiments were carried out at 37 0C.
IRBP removed retinol with approximately first order kinetics. The rate constant for all-trans retinol removal increased linearly with the concentration of IRBP, reaching 0.29±0.05 min-1 at an extracellular concentration of 20 µM. At the same extracellular concentrations of IRBP, removal of retinol proceeds with a higher rate constant from mouse than from frog rod photoreceptors. When a similar to the physiological 5 µM concentration of IRBP was present in the extracellular solution, from the time before bleaching and throughout the experiment, the concentration of retinol in the outer segment declined rapidly, having reached its peak immediately after bleaching.
IRBP is highly effective in removing the retinol generated in mouse rod outer segments after bleaching. The results indicate that the small diameter of the mammalian rod outer segment may be an important independent factor in facilitating the removal of retinol.
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