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。 光转导被认为是由cGMP的一过性减少所介导的 杆外段(ROS)上的水平线。这一过程的核心是 涉及视紫红质分子的光激活的酶级联反应， 称为转导蛋白和cGMP的调节性GTP结合蛋白 磷酸二酯酶(PDE)。转导蛋白和PDE的亚基已经纯化 并在功能上进行了重组。这为我们提供了一个学习的机会 分子水平上的激活机制。的结构和功能 这些亚基及其在光传导中的相互作用和调控 过程将通过生化和光谱技术进行研究。 我建议进行以下研究：(1)巯基的作用 光解视紫红质激活转导蛋白中的基团 采用化学改性方法进行研究。谷胱甘肽可能参与血管紧张素系统 我们将研究视觉兴奋的调节。(2)化学品 视紫质、转导蛋白和PDE的特定位点的修饰将是 对涉及的位置和氨基酸残基进行了调查 各种功能部位。(3)相互作用和相对接近 ROS中cGMP级联的成分将通过化学方法进行研究 用双官能团试剂和光亲和法研究交联剂 使用叠氮衍生的转导蛋白和PDE亚基进行交联。(4) 荧光能量转移将被用来绘制T的结构 转导蛋白的α亚基。GTP结合位点之间的距离， 特定的巯基、霍乱和百日咳毒素 将测量ADP-核糖化位点。T的构象变化 与GTP、ADP-核糖化和 化学修饰将通过圆二色谱(CD)进行研究。它 预计这个项目将促进我们对 了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.