Topography of rhodopsin in retinal rod outer segment disk membranes. Photochemical labeling with 1-azidopyrene

Abstract

1-Azido[3H]pyrene ([3H]AP) has been synthesized with high specific radioactivity (3 Ci/mmol) and used to photochemically label retinal rod outer segment disk membranes. The reagent reacts with rhodopsin and a Mr approximately 240000 protein as well as with membrane lipids. When [3H]AP-rhodopsin is digested with thermolysin in the disk membrane, both membrane-bound fragments of rhodopsin, F1 and F2, are found to contain [3H]AP. Reaction of the reagent appears to be restricted to the lipophilic surface of rhodopsin inasmuch as the presence of the nitrene scavenger glutathione in the aqueous medium does not significantly reduce 3H incorporation into rhodopsin. Labeled F1 and F2 were prepared, their cyanogen bromide peptides partially separated, and specific radioactivities determined. A factor of 4.4-fold in specific radioactivities of peptide pools was found, which suggests that some specificity has been shown in the reaction of [3H]AP toward different surfaces of rhodopsin.