THE CYCLIC GMP CASCADE OF RETINAL ROD CELLS

拟议研究的目标是了解 脊椎动物视网膜视杆细胞视觉兴奋的分子基础。 光转导被认为是通过cGMP的瞬时减少介导的 视杆外节（ROS）水平。 这一进程的核心是 光激活酶级联反应，涉及视紫红质分子， 一种称为转导素的调节性GTP结合蛋白和一种cGMP 磷酸二酯酶（PDE）。 转导素和PDE的亚基已被纯化 并进行功能性重组。 从而为我们提供了一个学习的机会 分子水平上的活化机理。 的结构和功能 这些亚基，它们在光转导蛋白中的相互作用和调节 过程将通过生物化学和光谱技术进行研究。 本人拟开展以下研究：（1）巯基的作用 在由光解的视紫红质激活转导蛋白中的基团将是 通过化学修饰研究。 谷胱甘肽可能参与了 将检查视觉兴奋的调节。 (2)化学 在视紫红质、转导素和PDE的特定位点上的修饰将是 进行探针的位置和氨基酸残基参与 各种功能场所。 (3)相互作用和相对接近 将通过化学方法研究ROS中cGMP级联的组分。 使用双功能试剂和通过光亲和性的交联研究 使用叠氮基衍生的转导素和PDE亚基进行交联。 （四） 荧光能量转移将用于绘制T 转导素α亚单位。 GTP结合位点之间的距离 霍乱和百日咳毒素的巯基 将测量ADP-核糖基化位点。 T的构象变化 与GTP结合、ADP核糖基化和 化学改性将通过圆二色性（CD）进行研究。 它 预计该项目将推进我们的知识， 理解视觉兴奋的分子机制。

The goal of the proposed research is to gain an understanding of the molecular basis of visual excitation in vertebrate retinal rod cells. Phototransduction is thought to be mediated by transient decrease of cGMP level on rod outer segments (ROS). Central to this process is a light-activated enzyme cascade which involves the rhodopsin molecule, a regulatory GTP binding protein called transducin and a cGMP phosphodiesterase (PDE). Subunits of transducin and PDE have been purified and functionally reconstituted. Thus provides us an opportunity to study the activation mechanism on molecular level. The structure and function of these subunits, their interaction and regulation in the phototransducin process will be investigated by biochemical and spectroscopic techniques. I propose to carry out the following studies: (1) The role of sulfhydryl groups in the activation of transducin by photolyzed rhodopsin will be studied by chemical modification. Possible involvement of glutathione in the regulation of visual excitation will be examined. (2) Chemical modification on specific sites of rhodopsin, transducin and PDE will be carried out to probe the location and amino acid residues involved in various functional sites. (3) Interaction and relative proximity of components of the cGMP cascade in ROS will be investigated by chemical cross-linking studies using bifunctional reagents and by photoaffinity cross-linking using azido-derivatized transducin and PDE subunits. (4) Fluorescence energy transfer will be used to map the architecture of the T Alpha subunit of transducin. Distances between GTP binding site, the specific sulfhydryl group, the cholera and pertussis toxins ADP-ribosylation sites will be measured. Conformational changes of the T Alpha subunit associated with the binding of GTP, ADP-ribosylation, and chemical modification will be investigated by circular dichorism (CD). It is anticipated that this project will advance our knowledge in understanding the molecular mechanism of visual excitation.