RETINAL CYCLIC GMP CASCADE

该项目的长期目标是阐明视觉的机制。 脊椎动物光感受器细胞的激发。具体目标集中在 转导蛋白的偶联作用，它是一种GTP结合蛋白，调节 从光激活视紫红质到cGMP的信息流 磷酸二酯酶。拟议中的研究是PERVICE的产物 从实验室获得的结果。(1)其分子结构 将对转导进行研究。特定化学物质的位置 将确定转导蛋白T-α亚单位上的修饰位点 通过多肽测序。这些站点之间的距离将通过以下方式估计 荧光能量传递测量及原位交联 实验。T-α的拓扑结构将基于 并与建议的三维模型进行比较 T-Alpha[Hingorani and Ho，FEBS Letter 220，15-22.(1987)]。(2)角色 我们将考察镁离子对T-α的耦合作用。MN2+将是 取代镁离子与结合部位的配位作用 Mn2+将通过电子自旋共振光谱进行研究 ~(17)O标记鸟嘌呤核苷酸与~(17)O富集水的相互作用 分子。(3)将尽最大努力阐明激活情况 CGMP磷酸二酯酶的作用机制。非抑制光亲和力 探针将连接到T-α-GPP(NH)p络合物和PGamma 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-α与T-α相互作用的部位 GPP(NH)；PGamma将通过交联法和肽图谱进行探测 学习。此外，还研究了其水动力性质的变化。 与PDE-T-α-GPP(NH)p络合物相关联的活化过程 通过沉淀物研究进行检验。结果应该提供一个 对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.