Quantitation of the effect of hydroxylamine on rhodopsin palmitylation.
Rhodopsin (the photosensitive rod visual pigment) has been a model for photobiologic studies of the opsins as well as a structural model for G-protein-coupled receptors. The two palmitate groups attached to cysteines 322 and 323 are thought to serve as membrane anchors for the rhodopsin C-terminus, but the absence of the palmitates does not alter membrane localization. However, removal of the palmitates affects rhodopsin function. Therefore, it is important to quantitate the stability of rhodopsin palmitates to hydroxylamine, which is a widely utilized reagent in biochemical preparations of the apoprotein. We have developed a mass spectrometric method to quantitate the resulting opsin palmitylation. Our data show that both of the bovine rhodopsin palmitates are labile to hydroxylamine, with significant depalmitylation occurring at concentrations of >or=100 mM, with an EC(50) of 220 mM L(-1). The palmitate at position 322 is the more stable to hydroxylamine. Samples prepared in the presence of >50 mM should therefore be considered to be at least partially depalmitylated and the results interpreted accordingly.
Jackson
Wesley
W
Storm Eye Institute, Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA.
Ablonczy
Zsolt
Z
Crouch
Rosalie K
RK
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D019811:Hydroxylamine / Q000494:pharmacology*; D054878:Lipoylation / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019308:Palmitic Acid / Q000378:metabolism*; D011487:Protein Conformation; D012243:Rhodopsin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism
D019811:Hydroxylamine; D019308:Palmitic Acid; D012243:Rhodopsin