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.
The presence of O-acetyl-5-methoxytryptophol in the pineal glands of rats kept in the dark for 8 h, but not in the light, has been shown by means of g.l.c.-mass spectrometry. It is suggested that this compound may be the biologically active precursor of circulating methoxytryptophol.
Purple membranes were isolated from Halobacterium halobium bleached and regenerated with all-trans-[15-3H]retinal. The incorporation of label was 1.2 mol of retinal/mol of bacterio-opsin. The [3H]retinyl-bacterio-opsin obtained from regeneration was hydrolysed to give tritiated retinyl-lysine, which, on hydrogenation to N-epsilon-perhydro[3H]retinyl-lysine and reaction with 1-fluoro-2,4-dinitrobenzene, gave bis-(2,4-dinitrophenyl)-N-epsilon-perhydro[3H]retinyl-lysine. This result confirmed that the retinyl moiety of the chromophore is attached to an epsilon-amino group of lysine.