May 2008
Volume 49, Issue 13
ARVO Annual Meeting Abstract  |   May 2008
Rhodopsin Palmitylation: Quantitation of the Effect of Hydroxylamine
Author Affiliations & Notes
  • R. K. Crouch
    Ophthalmology, Medical Univ of South Carolina, Charleston, South Carolina
  • Z. Ablonczy
    Ophthalmology, Medical Univ of South Carolina, Charleston, South Carolina
  • W. Jackson
    Ophthalmology, Medical Univ of South Carolina, Charleston, South Carolina
  • Footnotes
    Commercial Relationships  R.K. Crouch, None; Z. Ablonczy, None; W. Jackson, None.
  • Footnotes
    Support  NIH Grant EY04939, Research to Prevent Blindness and Foundation Fighting Blindness, Inc.
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 1276. doi:
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      R. K. Crouch, Z. Ablonczy, W. Jackson; Rhodopsin Palmitylation: Quantitation of the Effect of Hydroxylamine. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1276.

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

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Purpose: : Rhodopsin contains two palmitate groups at cysteines 322 and 323. These palmitates are known to have some lability to hydroxylamine but the effect of hydroxylamine on rhodopsin palmitates has not been quantified. Several studies have shown that removal of the palmitates affects various aspects of the function of protein. As hydroxylamine is routinely used in biochemical preparations of opsin for biochemical studies, loss of palmitates could affect the results. The purpose of this study was to quantitate the lability of the opsin palmitate groups, identify the primary site of removal of the palmitates and determine the level of palmitylation in native rhodopsin membranes.

Methods: : Light exposed bovine rod out segment preparations were incubated with hydroxylamine at varying concentrations. Analysis of palmitylation was by mass spectral analysis using HPLC/MS/MS and digestion by both CNBr and AspN.

Results: : Opsin palmitylation was found to be 1.97 palmitates/mole in the native membrane. About 3 % of the protein is singly palmitylated. On treatment of the membranes with hydroxylamine, significant depalmitylation occurred at concentrations of ≥0.05 mol/L, with an EC50 of 0.22 mol/L. The palmitate at position 323 is the more labile to hydroxylamine.

Conclusions: : Opsin samples prepared in the presence of ≥50 mM should therefore be considered to be at least partially depalmitylated and results interpreted accordingly.

Keywords: opsins • protein structure/function • protein modifications-post translational 

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