title: Labelling of the cytoplasmic domains of ovine rhodopsin with hydrophilic chemical probes.
abstract: 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.
mesh_terms: 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
chemical_list: 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