Biochemical aspects of rhodopsin

Methods have been developed for obtaining high yields of pure rod outer segments from frozen bovine retina. Proteolysis of these organelles was then studied using papain which did not affect the 500nm absorbance indicating that the microenvironment surrounding the prosthetic group 11-cis retinal had not altered sufficiently for thermal bleaching to take place. The analysis of the products of digestion was carried out using reductive and non reductive polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Untreated rod outer segments consisted of a single polypeptide chain rhodopsin molecule of 36,000 molecular .eight whereas extensively papain treated samples gave rise to the formation of three non covalently held fragments of 23,000, 13,000 and 6,000 molecular weight. To characterise these species the rod outer segments were variously labelled with 11-cis [15- 3H] retinal N-ethy1(-,3 1~iC7maleimide and j2Phosphato. After papain treatment all the labels remained associated with the membranes and electrophoresis showed that the three fragments of the digested protein were differential, labelled and that one of the three fragments consisted of three polypeptide chains held together by two disulphide bridges. The rhodopsin solecule had thus been cleaved into a non covalent complex which displayed many properties identical to the untreated protein.