RHODOPSIN TOPGRAPHY IN THE ROD CELL DISC MEMBRANE

我们建议为矢量方向和站点开发一个完整的图景 视紫红质在视紫红质视杆外段视盘膜的表面暴露。这 应该确定视紫红质是否是一种跨膜蛋白，并导致 测定其在膜中的侧面性。各种产品的本地化 视紫红质中的重要部位应该有助于构建 解释视紫红质在视觉传导中的作用模式。 将制备天然侧渗透完好的盘膜，并 特色化的。它们将被各种酶和化学试剂标记。 转谷氨酰胺酶将用于标记视紫红质与DNP-身体碱，以及 挂靠地点(S)另行确定。高反应性非穿透性 氮化物前体N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate将用于 确定视紫红质的额外多肽区域的表面暴露。 将制备针对视紫红质羧基末端区域1‘-34’的抗体。 抗体结合物将用于标记质膜中的视紫红质和 用于确定视紫红质表面定位的盘状膜 羧基末端。存在于膜脂中的视紫红质区域 环境将使用光反应试剂1-叠氮戊二烯进行标记。 将从视紫红质的疏水表面分离标记的多肽。

We propose to develop a complete picture for the vectorial orientation and sites of surface exposure of rhodopsin in the rod outer segment disc membrane. This should firmly establish whether rhodopsin is a transmembrane protein and lead to a determination of its sidedness in the membrane. Localization of various important sites in rhodopsin should assist in construction of theories to explain the mode of action of rhodopsin in visual transduction. Osmotically intact disc membranes of native sidedness will be prepared and characterized. They will be labeled with various enzymes and chemical reagents. Transglutaminase will be used to label rhodopsin with DNP-cadaverine, and the site(s) of attachment will be determined. The highly reactive nonpenetrating nitrene precursr N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate will be used to determine the surface exposure of additional peptide regions of rhodopsin. Antibodies to rhodopsin's carboxyl-terminal region 1'-34' will be prepared. Antibody conjugates will be used to label rhodopsin in the plasma membrane and disc membranes in order to determine the surface localization of rhodopsin's carboxyl terminal. The region of rhodopsin which exists in the membrane-lipid environment will be labeled using the photoreactive reagent 1-azidopyrene. Labeled peptides from rhodopsin's hydrophobic surface will be isolated.