Tryptophan in bovine rhodopsin: its content, spectral properties and environment.

The tryptophan content of purified bovine rhodopsin was obtained by two independent methods: direct analysis of hydrolysates prepared by digestion of opsin with methanesulfonic acid containing 0.2% 3-(2-aminoethyl)indole and a computer-assisted analysis of the near-UV spectrum of rhodopsin. Both methods gave a value of eight tryptophan residues per rhodopsin. Based on the near-UV spectral analysis, the light-induced difference spectrum of rhodopsin, and the susceptibility of residues to oxidation by N-bromosuccinimide, we concluded that approximately half of the tyrosine and tryptophan residues are shielded to some extent from the aqueous solvent, that two of the tryptophan residues are in very apolar environments, and that following light excitation at least one of these tryptophan residues and several tyrosines are exposed to an aqueous environment. Analysis of rhodopsin absorption in the far-UV indicated that below 240 nm, approximately half of the absorption is due to aromatic residues and that the other half is largely due to the peptide bond. The effect of illumination on secondary structure is to induce a loss in helical structure, calculated to involve 35% of the amino acid residues in purified rhodopsin. If light-induced changes in secondary structure are specifically excluded, most of these results can be extended to bovine rod outer segment membranes.

Structure of the carbohydrate moieties of bovine rhodopsin.

The sugar chains of bovine rhodopsin were released from the polypeptide moiety by hydrazinolysis and reduced with NaB[3H]4 after N-acetylation. The radioactive oligosaccharides thus obtained were fractionated into three components by paper chromatography. The structures of these components were elucidated as GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 (Man alpha 1 leads to 6)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(Man alpha 1 leads to 3 and 6 Man alpha 1 leads to 6)Man beta leads to 4GlcNAc beta 1 leads to 4GlcNAc, and GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(Man alpha 1 leads to 3 (Man alpha 1 leads to 6)Man alpha 1 leads to 6)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, by sequential exoglycosidase digestion, methylation analysis, and endo-beta-N-acetylglucosaminidase D digestion. The unusual features of the sugar chains of rhodopsin molecule seem to support the proposed processing pathway for the biosynthesis of asparagine-linked sugar chains of glycoproteins.

Synthesis of rhodopsin and opsin in vitro.

Isolated bovine retinas have been used to study the synthesis of rhodopsin and the renewal of photoreceptor rod outer segments. Both leucine and glucosamine served as radioactive tracers to follow rhodopsin synthesis. In both cases the rod outer segment preparations contained large amounts of labeled macromolecules chromatographically distinct from rhodopsin, the latter representing only about 10% of the high molecular weight labeled material. However, electrophoresis on polyacrylamide gels with sodium dodecyl sulfate indicated that as much as 60-68% of the radioactivity coincided with opsin, the apoprotein of rhodopsin. Treatment of labeled rod outer segments with 9-cis-retinal caused much of the non-rhodopsin label to be converted to isorhodopsin. After such treatment the fraction of the label in visual pigment rose from about 10 to 51% with leucine as the radioactive tracer and to 78% with glucosamine. Similar treatment of bleached outer segments labeled with leucine gave identical results with complete regeneration of isorhodopsin (lambda max 487 nm) which then accounted for 56% of the labeled macromolecules. No such conversion occurred in controls lacking 9-cis-retinal. Both 9-cis- and 11-cis-retinal were effective but all-trans-retinal was ineffective in producing the conversion. Under in vitro conditions opsin appears to be accumulated in the outer segment prior to the addition of retinal.