Topography of rhodopsin in rod outer segment disk membranes. Photochemical labeling with N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate

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Abstract
Rod cell disk membranes were photochemically reacted with the water-soluble, membrane-impermeable nitrene precursor N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonate [NAP-taurine, NAPT]. Rhodopsin, minor membrane proteins and lipids all incorporate the (nitrophenyl) [35S]taurine (NPT) label. Rhodopsin may be labeled in the dark using prephotolyzed NAPT. This reaction is presumably due to long-lived photoproducts of NAPT. NAPT modifies rhodopsin in the membrane in a selective manner; some cyanogen bromide peptides of NPT-rhodopsin contain appreciable NPT labeled and some contain essentially none. Precise specific radioactivities were not determined for the [35S]NPT-peptides since loss of label from some of the peptides was observed during purification procedures. Rhodopsin''s carboxyl-terminal cyanogen bromide peptides are well labeled when the protein is modified in disk membranes but the amino-terminal peptide is poorly labeled. When rhodopsin is labeled in reconstituted membranes in which both surfaces of rhodopsin are accessible to reagent, labeling of rhodopsin''s amino-terminal peptide is enhanced. Rhodopsin''s carboxyl-terminal region apparently is located at the cytoplasmic (externa) surface of disk membranes and its amino terminus is located at the intradiskal membrane surface.