Biochemistry of Visual Pigments: II. PHOSPHOLIPID REQUIREMENT AND OPSIN CONFORMATION FOR REGENERATION OF BOVINE RHODOPSIN

Abstract

Regeneration of digitonin-extracted rhodopsin at pH 6.0 was maximal (about 60%) in the presence of 1.3 moles of all-trans-retinal added per mole of rhodopsin. The regenerated pigment was identical with native rhodopsin in absorption and circular dichroism spectra. Rhodopsin in outer segments regenerated itself up to 30% and the addition of all-trans-retinal was without effect. Partial extraction of lipid from outer segments with petroleum ether resulted in a complete loss in regenerability. Regenerability was also lost by phospholipase A treatment of outer segments. The addition of the lipid extract or phospholipid to the partially delipidated outer segments fully restored regenerability. The results were interpreted as suggesting that phospholipid was required for the regeneration of the chromophore. Circular dichroism studies in the ultraviolet region on digitonin extracts and outer segments indicated that if rhodopsin was made susceptible to conformational change on photobleaching by phospholipase A treatment or petroleum ether extraction, it was no longer capable of regeneration. From these results, it was suggested that phospholipid might have an additional function in stabilizing the native conformation of opsin which would be essential for the regenerability of rhodopsin. Regenerability was found to be more sensitive to Emulphogene than opsin conformation. This was taken to suggest that there might be two separate phospholipids involved: one for the chromophore and the other for the native conformation of opsin.