The research has two long-term objectives: to elucidate the nature and physiological significance of interactions between visual pigment and G-protein in the vertebrate photoreceptor (Project 1); and to identify and characterize reactions that, in the dark-adapting eye, mediate the formation of 11-cis retinoid and the delivery of 11-cis retinal chromophore to opsin (Project 2). The Project 1 experiments, employing isolated retina and ROS preparations of skate and amphibian, will examine specific properties of G and the G-pigment interaction: (a) capacity of the Alpha and Beta subunits of G to bind, in light-dependent manner, to the pigment-containing ROS membranes (to be analyzed by extraction and electrophoretic isolation of G AlphaBeta); (b) capacity for ligand charging of G Alpha (to be analyzed through use of 32P-AAGTP, a photoaffinity probe); and (c) correlation of G binding/activation with state of phosphorylation of opsin, and binding of 48K protein to the ROS membranes. Conditions of irradiation to be used will be employed in parallel electrophysiological experiments, for quantitative comparison of the biochemical data with the behavior of receptor threshold. In related experiments, properties of G will be examined (in rat) following dietary deprivation of vitamin A, a treatment known to depress ERG b-wave sensitivity. Overall, the Project 1 studies are aimed at elucidating the molecular basis of sensitivity control during light/dark adaptation; information obtained may be especially useful for characterizing animal models expressing degeneration and/or abnormal development of the photoreceptors. Project 2: To gain information of the all-trans to 11-cis isomerization, H3-all-trans retinoids will be introduced subretinally into eyecup and intact eye preparations, and the fates of these substances examined after light/dark adaptation. Procedures will involve local injection of fluid containing vs. lacking the test retinoid, followed by sectioning of the tissue, and by extraction and HPLC analysis of retinoid products formed. Related experiments will examine effects, on isomerization and rhodopsin regeneration, of other test substances introduced subretinally, e.g., interstitial retinol-binding protein (IRBP). Beyond immediate significance for vitamin A physiology, the Project 2 studies should provide data (and methodology) of direct interest to others studying interactions between the retina and pigment epithelium.
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