[Visual rhodopsin. III. Complete amino acid sequence and topography in a membrane].

Tryptic hydrolysis of apomembranes, BNPS-skatole cleavage of carboxymethylated rhodopsin and thermolytic digestion of native membranes were carried out to obtain the peptides necessary for the polypeptide chain reconstruction. Gel-filtration on Bio-Gel P-30 in 80% formic acid, ion-exchange and reversed-phase high performance liquid chromatography were used for the peptide isolation. A comparison of rhodopsin hydrophobicity profile with the accessibility of the polypeptide chain in native photoreceptor membranes for proteases allowed to distinguish seven alpha-helical segments and propose a model for arrangement of the protein molecule in the membrane.

[Primary structure of rhodopsin. II. Chymotrypsin hydrolysis peptides].

Apomembranes prepared from the photoreceptor disks were subjected to chymotryptic hydrolysis. The insoluble material, containing the membrane-bound peptides was removed by centrifugation, and the water-soluble peptides of the supernatant were separated by ion-exchange chromatography on AG 50W X 4 followed by high performance liquid chromatography. The insoluble peptides were separated by gel-filtration on Bio-Gel P-30 in 80% formic acid.

[Primary structure of rhodopsin. I. Cyanogen bromide degradation peptides].

Carboxymethylated bovine rhodopsin was subjected to cyanogen bromide cleavage at methionine residues. The resultant products were fractionated into the two groups according to the solubility of peptides in 2 M guanidine hydrochloride. Gel-filtration on Bio-Gel P-30 in 80% formic acid of each group followed by rechromatography and high performance liquid chromatography resulted in 15 peptides embracing the whole polypeptide chain of rhodopsin.

[Study of the molecular organization of visual rhodopsin in photoreceptor membranes by limited proteolysis].

Proteolysis of rhodopsin in disc membranes of right-side out orientation by thermolysin, papain and St. aureus V8 protease allowed to identify two highly exposed regions of polypeptide chain located on the cytoplasmic membrane surface: carboxyl terminal sequence 321-348 and the fragment 236-241. Incubation with chymotrypsin reveals the third site on the cytoplasmic surface, 146-147, accessible to proteolytic enzymes.