RHODOPSIN TOPGRAPHY IN THE ROD CELL DISC MEMBRANE

我们建议制定一个完整的图片矢量方向和网站 视紫红质表面暴露于视杆外节盘膜。 这 应该确定视紫红质是否是一种跨膜蛋白， 确定其在膜中的侧性。 各种本地化 视紫红质中的重要位点应该有助于理论的构建， 解释视紫红质在视觉传导中的作用方式。 将制备具有天然侧性的渗透完整椎间盘膜， 表征了 它们将被贴上各种酶和化学试剂的标签。 转氨酶将用于用DNP-尸胺标记视紫红质， 将确定连接部位。 高活性非渗透性 N-（4-叠氮基-2-硝基苯基）-2-氨基乙基磺酸氮烯酯将用于 确定视紫红质的另外的肽区域的表面暴露。 将制备针对视紫红质羧基末端区域1 '-34'的抗体。 抗体缀合物将用于标记质膜中的视紫红质， 盘膜，以确定视紫红质的表面定位 羧基末端 视紫红质存在于膜脂中的区域 将使用光反应试剂1-叠氮芘标记环境。 将从视紫红质的疏水表面分离标记的肽。

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.