May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Protein Complex Components of RGR Opsin Isolated From the Bovine Retinal Pigment Epithelium
Author Affiliations & Notes
  • P. Chen
    Ophthalmology, Doheny Eye Institute, Los Angeles, CA
  • H. Kochounian
    Molecular Immunology and Microbiology, Keck School of Medicine of the University of Southern California, Los Angeles, CA
  • H.K. W. Fong
    Ophthalmology, Molecular Immunology and Microbiology, Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA
  • Footnotes
    Commercial Relationships  P. Chen, None; H. Kochounian, None; H.K.W. Fong, None.
  • Footnotes
    Support  NIH Grants EY08364 and EY03040; Research to Prevent Blindness Foundation.
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 5127. doi:
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      P. Chen, H. Kochounian, H.K. W. Fong; Protein Complex Components of RGR Opsin Isolated From the Bovine Retinal Pigment Epithelium . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5127.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: To identify candidate proteins that form a structural and functional complex with the RGR opsin. Methods: Bovine RGR was isolated from RPE microsomes by an immunoaffinity procedure under various buffer conditions. RGR and co–purified proteins were separated by 12% SDS–PAGE and visualized with Coomassie Brilliant Blue and silver staining. The proteins in excised gel bands were reduced and alkylated by treatment with dithiothreitol and iodoacetamide, respectively. The samples were digested with trypsin at 30°C overnight, and the peptide fragments were analyzed by mass spectrometry on an LC/MS system. Correlating the MS/MS spectra to known protein sequence using the Sequest database search program identified the proteins of interest. Results: A number of proteins (P110, P100, P80, P65, P34 and P32) were co–isolated with RGR by immunoaffinity chromatography using different buffer conditions. Individual protein bands were affected by the presence of glycerol, salt concentration, detergent type, and staining method. Previous results identified P32 as 11–cis–retinol dehydrogenase. Other proteins were identified as Grp78 (P80) and RPE65 (P65). RPE65 was also detected consistently by Western blot analysis. Weaker bands remain to be identified. Conclusions: A few proteins co–purify consistently with RGR under various conditions and may form a protein complex with the RGR opsin. 11–cis–Retinol dehydrogenase and RPE65 are important visual cycle proteins that may interact with RGR in vivo. Grp78 may act to maintain normal structure and function of an RGR protein complex.

Keywords: protein purification and characterization • retinal pigment epithelium 
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