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RK Crouch, Z Ablonczy, R Darrow, DR Knapp, DT Organisciak; Loss Of Light-dependent Rhodopsin Phosphorylation In Rats Exposed To Intense Visible Light . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4542.
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Purpose: Phosphorylation of light activated rhodopsin is critical for the deactivation of the visual transduction process. The effect of the duration of light exposure at various times in the 24 h circadian cycle on rhodopsin phosphorylation was examined. Methods: SD rats were exposed to 1/2, 4, or 8 h of continuous 1500 lux light starting at 1 a.m. or 5 p.m. ROS was isolated immediately afterward in the presence of phosphatase inhibitors and later treated with AspN enzyme for the removal of the rhodopsin C termini. The phosphorylation of the peptides was analyzed by electrospray ionization tandem mass spectrometry coupled online to HPLC. Results: (1) Rhodopsin from rats exposed to intense visible light contains elevated levels of multiple phosphorylations. In samples exposed for 1/2 h, all the possible sites were phosphorylated. The amount of mono-, di-, and triphosphorylation was elevated and comparable, while tetra- and penta- and hexaphosphorylation was observed at only slightly above the baseline levels. (2) The amount and multiplicity of phosphorylation was reduced in the samples exposed for 4 h and further reduced after 8 h. (3) The stage of the circadian rhythm at the start of the light exposure affected phosphorylation. These effects were most pronounced after 1/2 h and diminished by 8 h. Conclusions: (1) The reduction in the amount of phosphorylation is probably due to a reduction in the rhodopsin to opsin ratio. The production and transfer of the 11-cis retinal chromophore may be rate limiting in opsin regeneration. (2) There is a pronounced difference between the activity of the kinase acting on two sets of three phosphorylation sites. The kinase may differentiate between the three serine and three threonine sites. (3) The production of the chromophore may be affected by factors dependent on the circadian rhythm.
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