Labelling of the cytoplasmic domains of ovine rhodopsin with hydrophilic chemical probes.
The disposition of polypeptide chain of ovine rhodopsin in the photoreceptor disc membrane was investigated by using two hydrophilic reagents, 3,5-di-[125I]iodo-4-diazobenzenesulphonate [( 125I]DDISA) and [14C]succinic anhydride. Both reagents were used to modify rhodopsin in intact disc membranes under conditions where no loss of A500 occurred. Reaction of [125I]DDISA with rhodopsin approached completion after 30 min. Binding was saturated at a 75-fold molar excess of reagent, which gave binding ratios of up to 2 mol/mol of rhodopsin. Proteolysis of rhodopsin, using Staphylococcus aureus V8 proteinase, yielded two membrane-bound fragments, both of which contained bound radioactive probe. Subsequent CNBr cleavage of these fragments produced five radiolabelled peptides which corresponded to the C-terminal region and cytoplasmic loops of rhodopsin. Similar studies with [14C]-succinic anhydride also gave binding ratios of up to 2 mol/mol of rhodopsin. Sequencing of the [14C]succinylated peptides identified the location of the reactive sites as lysine residues 66, 67, 141, 245, 248, 311, 325 and 339 in the polypeptide chain. Non-permeability of both probes was demonstrated by the absence of any radioactivity associated with the intradiscal N-terminal glycopeptide. Sonication of membranes in the presence of [125I]DDISA led to the incorporation of label in this peptide.
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000032:analysis; D002850:Chromatography, Gel; D003488:Cyanogen Bromide; D003593:Cytoplasm / Q000032:analysis; D003979:Diazonium Compounds; D010450:Endopeptidases; D008958:Models, Molecular; D010446:Peptide Fragments / Q000032:analysis; D010786:Photoreceptor Cells / Q000032:analysis*; D012168:Retinal Pigments; D012243:Rhodopsin; D012697:Serine Endopeptidases; D012756:Sheep; D013386:Succinates; D013387:Succinic Anhydrides
D003979:Diazonium Compounds; D010446:Peptide Fragments; D012168:Retinal Pigments; D013386:Succinates; D013387:Succinic Anhydrides; C007308:diazodiiodosulfanilic acid; C031801:succinic anhydride; D012243:Rhodopsin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D003488:Cyanogen Bromide