THE RETINAL CYCLIC GMP CASCADE

该项目的长期目标是阐明视觉的机制， 脊椎动物感光细胞的兴奋。 具体目标侧重于 转导素的偶联作用，一种调节细胞凋亡的GTP结合蛋白， 从光敏视紫红质到cGMP的信息流 磷酸二酯酶 拟议中的研究是以前的研究成果。 从实验室获得的结果。 (1)的分子结构 将研究转导素。 特定化学品的位置 将鉴定转导素的T-α亚基上的修饰位点 通过肽测序。 这些站点之间的距离将通过 荧光能量转移测量和原位交联 实验 T-alpha的拓扑结构将基于 这些测量结果，并与建议的三维模型进行比较， T-alpha [Hingorani和Ho，FEBS Letter 220，15-22。（1987）]。 (2)的作用 将检查Mg 2+对偶联作用T-α的影响。 Mn 2+将 取代到Mg 2+结合位点和结合的配位 Mn 2+将通过电子自旋共振光谱法通过 与17 O标记的鸟嘌呤核苷酸和17 O富集水的相互作用 分子。 (3)一个主要的努力将投入到阐明激活 cGMP磷酸二酯酶的作用机制。 非抑制性光亲和性 探针将连接到T-α-Gpp（NH）p复合物和P γ-Gpp（NH）p复合物上， 磷酸二酯酶的亚基，将这些蛋白质转化为 光亲和标记分子。 T-alpha和T-alpha之间的相互作用位点 Gpp（NH）;和Pgamma将通过交联和肽图谱进行探测 问题研究 此外，还研究了在不同的水动力条件下， 与活化过程相关的PDE-T-alpha-Gpp（NH）p复合物 通过沉降研究进行检查。 结果应该提供一个 对细胞信号转导分子机制的认识 GTP结合蛋白。

The long term goal of the project is to elucidate the mechanism of visual excitation in vertebrate photoreceptor cells. The specific aim focuses on the coupling action of transducin, a GTP-binding protein that regulates the flow of information from photo-activated rhodopsin to the cGMP phosphodiesterase. The proposed studies are an outgrowth of pervious results obtained from the laboratory. (1) The molecular structure of transducin will be investigated. The locations of specific chemical modification sites on the T-alpha subunit of transducin will be identified by peptide sequencing. Distances between these sites will be estimated by fluoresence energy transfer measurements and by in situ cross-linking experiments. A topological structure of T-alpha will be constructed based on these measurements and compared to a proposed three-dimensional model of T-alpha [Hingorani and Ho, FEBS Letter 220, 15-22.(1987)]. (2) The role Mg2+ on the coupling action T-alpha will be examined. Mn2+ will be substituted into the Mg2+ binding site and the coordination of the bound Mn2+ will be investigated by electron spin resonance spectroscopy via the interactions with 17O labeled guanine nucleotides and 17O-enriched water molecules. (3) A major effort will be put into elucidating the activation mechanism of the cGMP phosphodiesterase. Non-inhibitory photoaffinity probes will be attached to the T-alpha-Gpp(NH)p complex and the Pgamma subunit of phosphodiesterase, transforming these proteins into a photoaffinity labeling molecule. The interacting sites between T-alpha- Gpp(NH); and Pgamma will be probed by cross-linking and peptide mapping studies. Furthermore, the changes of the hydrodynamic properties of the PDE-T-alpha-Gpp(NH)p complex associated with the activation process will be examined by sedimentation studies. The results should provide an understanding on the molecular mechanism of signal transduction mediated by GTP-binding proteins.