May 2007
Volume 48, Issue 13
ARVO Annual Meeting Abstract  |   May 2007
Release of 11-cis-Retinal From CRALBP
Author Affiliations & Notes
  • J. C. Saari
    Ophthalmology, University of Washington, Seattle, Washington
  • G. G. Garwin
    Ophthalmology, University of Washington, Seattle, Washington
  • M. Nawrot
    Ophthalmology, University of Washington, Seattle, Washington
  • Footnotes
    Commercial Relationships J.C. Saari, None; G.G. Garwin, None; M. Nawrot, None.
  • Footnotes
    Support NIH Grant EY02317, EY01730, Research to Prevent Blindness, Inc
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 3257. doi:
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    • Get Citation

      J. C. Saari, G. G. Garwin, M. Nawrot; Release of 11-cis-Retinal From CRALBP. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3257.

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

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Purpose:: To develop assays for the release of 11-cis-retinal from cellular retinaldehyde-binding protein (CRALBP) and to explore conditions leading to release of the retinoid.

Methods:: 9-cis-retinal (used in place of 11-cis-retinal) was added to human recombinant CRALBP (rCRALBP) and excess retinoid removed by Ni-chromatography. Assays developed exploited the protection of the carbonyl of 9-cis-retinal from reaction with water-soluble carbonyl reagents when the retinoid is bound to native CRALBP. One assay followed the change in absorption when bound 9-cis-retinal (410 nm max abs) was released from CRALBP and reacted with NH2OH to form 9-cis-retinal oxime (355 nm max abs). A second assay employed HPLC to quantify the amount of retinal oxime formed upon incubation of CRALBP·9-cis-retinal with various RPE extracts and agents in the presence of 2 mM NH2OH.

Results:: After 3 hours of incubation of CRALBP·9-cis-retinal with NH2OH at room temperature and pH 7, 90% of the chromophore was recovered as 9-cis-retinal by HPLC analysis, demonstrating the stability of native CRALBP. Addition of 50% ethanol, which denatured the protein and released the retinoid, resulted in the detection of >95% 9-cis-retinal oxime. Urea (4 M) resulted in a perturbation of the ligand-binding domain as evidenced by the slow release of 9-cis-retinal (t1/2 12 min). Incubation of CRALBP·9-cis-retinal with subcellular fractions from bovine RPE homogenates did not release the retinoid. Lowering the pH from 7 to 5 resulted in a pronounced blue shift in the absorbance (pKa ~ 6), indicating that the ligand-binding domain had been perturbed.

Conclusions:: CRALBP is a water-soluble protein found in relatively high abundance in RPE and Muller cells. Based on in vitro enzymology, studies of CRALBP knockout mice, and human mutations, it is likely that apo-CRALBP accepts 11-cis-retinol from the isomerohydrolase reaction in the visual cycle and facilitates its oxidation to 11-cis-retinal. If this mechanism is correct, 11-cis-retinal must then be released from CRALBP. In this study we demonstrate that 11-cis-retinal is released from CRALBP at the approximate pH of an early endosome.

Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium 

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