Structural studies on membrane-bound bovine rhodopsin.
Our previous work has shown that the treatment of bovine rhodopsin with the proteolytic enzyme papain gives rise to a cleaved, but fully functional, complex consisting of three fragments, H, M and L (heavy, medium and light), held together by strong non-covalent forces. By using some of the chemical and physical differences between the three fragments, a protocol for the preparative isolation of each fragment was devised. Purified M-fragment, which had been radiochemically labelled at the retinal-binding site was treated with CNBr and the mixture subjected to a multi-step separation to furnish a retinyl peptide. The sequence analysis of the latter showed that the retinal-binding lysine residue was located at position 296 from the N-terminal of rhodopsin (or residue 53 from the C-terminal). In order to ascertain the position of the cytoplasmic loop which exists between the M- and L-fragments, radiochemically labelled L-fragment was isolated from the cleaved complex. The purified L-fragment was shown to consist of two populations of peptides which were produced by the action of papain on the bonds between Lys-311 and Gln-312 and between Gln-312 and Phe-313.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis / Q000302:isolation & purification; D012168:Retinal Pigments / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000032:analysis
D010446:Peptide Fragments; D012168:Retinal Pigments; D012243:Rhodopsin; D010206:Papain