VISUAL PIGMENT AND PHOTORECEPTOR ADAPTATION

这项研究有两个长期目标：阐明 视色素与视网膜色素相互作用的生理意义 脊椎动物光感受器中的G蛋白（项目1）;并鉴定和 表征暗适应眼中介导 11-顺式类维生素A的形成和11-顺式视网膜发色团的递送 视蛋白（Project 2）。 项目1实验，采用离体视网膜和ROS制剂 的溜冰鞋和两栖动物，将检查具体性质的G和 G-色素相互作用：（a）G的α和β亚基与G-色素相互作用的能力。 以光依赖性方式结合到含有色素的ROS膜上 (to通过提取和电泳分离G α β）;（B）G α的配体充电能力（待分析 通过使用32 P-AAGTP，一种光亲和探针）;和（c）G 与视蛋白的磷酸化状态的结合/活化，以及 48 K蛋白的活性氧膜。 使用的辐照条件 将用于并行电生理学实验， 生化数据的定量比较， 受体阈值 在相关的实验中，G的性质将是 在饮食剥夺维生素A（一种治疗）后检查（大鼠） 已知会降低ERG b波敏感性 总体而言，项目1研究 目的在于阐明敏感性控制的分子基础， 光/暗适应;获得的信息可能特别有用， 表征表达变性和/或异常的动物模型 光感受器的发展。 项目2：获得全反式到11-顺式异构化的信息， H3-全反式类维生素A将在视网膜下引入眼杯， 完整的眼准备，以及这些物质在 光/暗适应 程序将涉及局部注射液体 含有与缺乏测试类维生素A，然后切片的 组织，并通过提取和HPLC分析所形成的类维生素A产物。 相关的实验将检查影响，异构化和视紫红质 再生，视网膜下引入的其他测试物质，例如， 间质视黄醇结合蛋白（IRBP）。 超越眼前的意义 对于维生素A生理学，项目2研究应提供数据（以及 方法学），其他研究人直接感兴趣， 视网膜和色素上皮。

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.