| The biochemical basis of vision in the vertebrate eye has been actively studied for more than a century. While much progress has been made, the key reaction for the formation of active visual pigments has remained unsolved. In this thesis, the "missing reaction" of the vertebrate visual cycle, the energy-dependent isomerization of all-trans-retinoids to 11-cis-retinoids, is investigated through biochemical and pharmacological means.;The biochemical processes of vision are first probed through the use of 1,5-di-(p-aminophenoxy)pentane (DAPP), the most powerful and selective pharmacological inhibitor of dark adaptation known. It is shown that this drug inhibits rhodopsin regeneration by preventing formation of all 11-cis-retinoids in the eye and by running down stores of 11-cis-retinyl palmitate. Based on structure/activity studies and other experiments, it is concluded that DAPP and its analogs cause night blindness by short-circuiting the visual cycle when they catalyze the isomerization of 11-cis-retinal back to all-trans-retinal.;Next, the biochemical pathway of isomerization of retinoids in living animals is examined through double-label radioisotope studies in normal animals and in animals treated with DAPP or other inhibitors of dark adaptation. From these studies, it is possible to conclude that isomerization of all-trans-retinoids to 11-cis-retinoids in the living eye occurs at the alcohol oxidation state.;Finally, a novel retinoid isomerase activity was discovered in homogenates of frog retina/pigment epithelium that catalyzes the endergonic isomerization of all-trans-retinoids to 11-cis-retinoids in darkness. The activity is heat-labile, membrane-bound, and localized primarily to the pigment epithelium in amphibians. Its activity is destroyed by low concentrations of detergents or ethanol. In partially purified preparations, added (11,12-$sp3$H) -all-trans-retinol is converted to 11-cis-retinol and other 11-cis-retinoids, while added labeled all-trans-retinal and all-trans-retinyl palmitate are not isomerized to a significant extent. It is likely that this enzyme system that completes the visual cycle is very important in the physiology and pathophysiology of vision. |
