MECHANISM OF VISUAL EXCITATION

我们的长期目标是阐明生物化学和生物物理 视觉兴奋和光感受器代谢的潜在机制。我们的 方法是在几个组织中解决重要的、相关的问题 感光细胞的水平。这些层次首先是视觉上的 颜料本身，特别是鸡锥形颜料，除了 建立它们的氨基酸序列，我们计划对其进行纯化和研究 色素的生物物理学(振动光谱，漂白动力学)。第二, 在光对色素的作用水平上，我们将寻求学习 原生光子能量存储的本质 光产物、底紫红质和变视紫质II的性质 使其能够与GTP结合蛋白相互作用并激活， 换能器。三是感光细胞膜的水平。我们想要 光感受器表面电位的测量与控制研究 膜，因为表面电势将对 带电底物的浓度(例如cGMP)、辅助因素(例如GRP)和 光感受器膜表面附近的酶(如转导蛋白) 激活和一些随后的酶事件发生。我们还计划 澄清膜中视紫红质的拓扑结构并将其提纯 锥形感光细胞膜。第四，是水平感光细胞， 尤其是在换能器本身，以及 视网膜细胞内的传递器和其他关键小分子。这里 我们将利用新的方法来研究转导蛋白(拉曼差值 光谱、阳离子结合、Meta II引发的构象变化)和 31P核磁共振波谱在细胞内研究中的应用 体内的递质和代谢物。这种集成的方法将提供 不仅对视觉兴奋和视网膜控制有了新的见解 新陈代谢，也导致了新的和更敏感的方式的发展 检测体内视网膜的健康和代谢状态。

Our long term goal is to elucidate the biochemical and biophysical mechanisms underlying visual excitation and photoreceptor metabolism. Our approach is to attack important, related problems at several organizational levels of the photoreceptor cell. These levels are first, the visual pigments themselves, especially chicken cone pigments where, besides establishing their amino acid sequences, we plan to purify and study the pigments biophysically (vibrational spectra, bleaching kinetics). Second, at the level of the action of light on the pigments, we will seek to learn the nature of the storage of the photon's energy by the primary photoproduct, bathorhodopsin, and the properties of metarhodopsin II which allows it to interact with and activate the GTP binding protein, transducin. Third is the level of the photoreceptor membrane. We want to measure and study the control of the surface potential of photoreceptor membranes, since the surface potential will have profound effects on the concentration of charged substrates (e.g. cGMP), co-factors (e.g. GRP), and enzymes (e.g. transducin) near the surface of photoreceptor membranes where activation and some subsequent enzymatic events takes place. We also plan on clarifying the topology of rhodopsin in the membrane and will purify cone photoreceptor membranes. Fourth, is the level photoreceptor cell, especially with regard to the transduction machinery itself, and the transmitters and other key small molecules within the retinal cells. Here we will utilize new approaches to transducin (Raman difference spectroscopy, cation binding, meta II initiated conformational changes) and the use of phosphorus 31 NMR spectroscopy to study intracellular transmitters and metabolites in vivo. This integrated approach will offer not only new insight into visual excitation and the control of retinal metabolism, but also lead to the development of new and more sensitive ways to assay the health and metabolic state of the retina in vivo.