Rhodopsin chromophore exchanges among opsin molecules in the dark.

Turnover of rhodopsin chromophore in vertebrate visual cells has been explored by light microscope autoradiography (LMARG) and radiobiochemical techniques. Retinol-binding protein (RBP) was isolated from human serum, its native ligand removed and replaced with [3H]-retinol. After reconstitution, [3H]-retinol-RBP was reassociated with prealbumin (PA), and the protein complex injected intravenously into dark-adapted animals. After selected intervals in the dark, animals were killed, and ocular tissues dissected under infrared illumination. Eyecups from frogs and mice were fixed (4 C) and after in situ reduction of the chromophore-protein linkage of rhodopsin with borane dimethyl amine (BDMA), processed histologically to retain lipids, or alternatively to extract them with chloroform-methanol (C-M), and LMARG performed. Rhodopsin was purified from detergent-solubilized mouse retinas by Concanavalin A (Con A) affinity chromatography and analyzed for radioactivity. Autoradiographic labeling of frog rod outer segments (ROS) was first detectable at 1 day postinjection, increasing over the duration of the experiment. At all times, label was distributed throughout the organelle in a diffuse pattern, although in certain cases a band of silver grains was also evident at the proximal end of the ROS, the site of new membrane assembly. Similar autoradiographic patterns were noted in mouse rods, although the kinetics of labeling differed in certain respects. In biochemical experiments, incorporation of [3H]-retinol into mouse rhodopsin was seen to occur very rapidly (less than 30 min), without an appreciable lag period. We interpret the diffuse labeling of ROS to result from an exchange in the dark of [3H]-vitamin A aldehyde for unlabeled opsin-bound chromophore, whereas the formation of a reaction band no doubt reflects the continual renewal of ROS membrane occurring in the dark. With respect to the former, the turnover of chromophore qualitatively resembles that found for membrane fatty acids.