May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Light Exposure Alters Carboxyethylpyrrole Modified Proteins in Rod Outer Segments
Author Affiliations & Notes
  • D. T. Organisciak
    Petticrew Research Laboratory, Biochemistry & Molecular Biology, Wright State University, Dayton, Ohio
  • R. M. Darrow
    Petticrew Research Laboratory, Biochemistry & Molecular Biology, Wright State University, Dayton, Ohio
  • L. S. Barsalou
    Petticrew Research Laboratory, Biochemistry & Molecular Biology, Wright State University, Dayton, Ohio
  • K. Renganathan
    Ophthalmic Research, Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio
  • J. S. Crabb
    Ophthalmic Research, Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio
  • J. W. Crabb
    Ophthalmic Research, Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio
  • Footnotes
    Commercial Relationships  D.T. Organisciak, None; R.M. Darrow, None; L.S. Barsalou, None; K. Renganathan, None; J.S. Crabb, None; J.W. Crabb, None.
  • Footnotes
    Support  NEI EY-14239, EY 15638, Foundation Fighting Blindness, Research to Prevent Blindness, BRTT 05-29 from the State of Ohio, the Ohio Lions Research Foundation and M. Petticrew, Springfield, OH.
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 153. doi:
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      D. T. Organisciak, R. M. Darrow, L. S. Barsalou, K. Renganathan, J. S. Crabb, J. W. Crabb; Light Exposure Alters Carboxyethylpyrrole Modified Proteins in Rod Outer Segments. Invest. Ophthalmol. Vis. Sci. 2008;49(13):153.

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Abstract

Purpose: : Oxidation of docosahexaenoic acid (DHA) can lead to the formation of carboxyethylpyrrole (CEP) protein adducts. To better understand light-induced protein modifications in retinal light damage we probed rod outer segment (ROS) proteins for CEP adducts after light exposure of varying duration and intensity.

Methods: : ROS were isolated from 2 or 3 groups of 4 rats on separate days by sucrose density ultracentrifugation. Samples were prepared from dark adapted rats at 4 hour intervals during the day and night, immediately after intense light exposure, and at various times after light treatment. To prevent in vitro oxidation a buffer containing 2mM DTPA, 100 µM BHT and a cocktail of proteolytic inhibitors was used. The buffer was purged with argon and all samples were stored under argon in liquid N2 until use. Following electrophoresis and western analysis, CEP positive bands were excised from the gels, digested with trypsin and proteins identified by mass spectroscopy.

Results: : CEP immunoreactivity in ROS isolated from dark adapted rats was present in a series of discrete electrophoretic bands, with relatively uniform staining over the day and night. A prominent band of ROS proteins in the 35-39 kDa range was found to contain transducin, glycolytic enzymes and perpherin. CEP adducts of tublin, actin, enolase and phosphoglycerate kinase were present in a 45-50 kDa molecular weight band. Following intense light exposure, CEP reactivity in ROS changed, primarily due to a decrease in staining for the 35-39 kDa proteins, while CEP-protein immunoreactivity in the 45-50 kDa range increased. These changes were light duration and intensity dependant and correlated with differences in transducin and arrestin immunoreactivity. Following light exposure ROS-CEP immunoreactivity and transducin staining increased with time in darkness; arrestin staining decreased. Total retinal CEP protein adduct formation was less affected by light or by subsequent darkness.

Conclusions: : The relatively uniform appearance of CEP staining in ROS during the day and night indicates that protein-lipid adduct formation is a constant and on going process. Intense light exposure changes the labeling of some CEP reactive ROS proteins in a manner consistent with the light-induced translocation of transducin and arrestin. The reappearance of ROS-CEP immunoreactivity with time in darkness suggests that oxidatively modified protein adducts may also be capable of translocation within photoreceptors.

Keywords: photoreceptors • oxidation/oxidative or free radical damage • retina 
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