Accessibility of the carbohydrate moiety of membrane-boound rhodopsin to enzymatic and chemical modification.
Galactose was specifically inserted into the carbohydrate moiety of rhodopsin by incubating retinal disk membranes with UDP-galactose: N-acetylglucosamine galactosyltransferase. The stoichiometry of labeling ranged from 1.2 to 1.8 (average = 1.5) residues of galactose per molecule of rhodopsin, indicating that some or all of the oligosaccharide chains of membrane-bound rhodopsin are readily accessible to enzymatic modification. These modified membranes were treated with galactose oxidase to generate an aldehyde at the C-6 position of the inserted galactose units. The enzymatically-oxidized membranes were then reacted with dansyl hydrazide to yield a fluorescent hydrazone which is sufficiently stable to permit spectroscopic analysis. This procedure for the specific attachment of a spectroscopic probe should be applicable to a wide variety of membrane glycoproteins.
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003619:Dansyl Compounds; D005690:Galactose / Q000032:analysis; D005692:Galactose Oxidase / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism; D006834:Hydrazines; D010786:Photoreceptor Cells / Q000378:metabolism*; D011485:Protein Binding; D012168:Retinal Pigments / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D003619:Dansyl Compounds; D006834:Hydrazines; D012168:Retinal Pigments; D012243:Rhodopsin; D005692:Galactose Oxidase; D005700:Galactosyltransferases; D005690:Galactose