Molday L L, Cook N J, Kaupp U B, Molday R S
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
J Biol Chem. 1990 Oct 25;265(30):18690-5.
A 240-kDa protein exhibiting immunochemical cross-reactivity with red blood cell spectrin has been shown to be directly associated with the 63-kDa cGMP-gated channel of bovine rod outer segments. When detergent-solubilized, chromatographically purified channel preparations were treated with Sepharose beads coupled to either an anti-240-kDa monoclonal antibody (PMs 4B2) or an anti-63-kDa channel monoclonal antibody (PMc 1D1), both the 240-kDa protein and the 63-kDa channel protein were concomitantly immunoprecipitated as analyzed by Western blotting of sodium dodecyl sulfate gels. Both of these antibody-Sepharose matrices also removed cGMP-gated channel activity as measured by functional reconstitution. In control studies anti-rhodopsin monoclonal antibody (Rho 1D4)-Sepharose beads removed residual rhodopsin, but not the 63/240-kDa complex or channel activity. Western blotting of purified rod outer segment disk and plasma membrane fractions and immunogold-dextran labeling of lysed rod outer segments indicated that the 240-kDa polypeptide, like the 63-kDa channel, is preferentially localized to the plasma membrane as visualized by electron microscopy. The 240-kDa protein does not appear to be directly involved in the cGMP-gated channel activity, but it may be part of a cytoskeletal system that serves to maintain the organization of the 63-kDa channel complex within the rod outer segment plasma membrane.
一种与红细胞血影蛋白具有免疫化学交叉反应性的240 kDa蛋白质已被证明与牛视杆外段的63 kDa cGMP门控通道直接相关。当用去污剂溶解并经色谱纯化的通道制剂用偶联有抗240 kDa单克隆抗体（PMs 4B2）或抗63 kDa通道单克隆抗体（PMc 1D1）的琼脂糖珠处理时，通过十二烷基硫酸钠凝胶的蛋白质印迹分析，240 kDa蛋白质和63 kDa通道蛋白都被同时免疫沉淀。通过功能重建测量，这两种抗体 - 琼脂糖基质也去除了cGMP门控通道活性。在对照研究中，抗视紫红质单克隆抗体（Rho 1D4） - 琼脂糖珠去除了残留的视紫红质，但没有去除63/240 kDa复合物或通道活性。纯化的视杆外段盘和质膜组分的蛋白质印迹以及裂解的视杆外段的免疫金 - 葡聚糖标记表明，240 kDa多肽与63 kDa通道一样，通过电子显微镜观察优先定位于质膜。240 kDa蛋白质似乎不直接参与cGMP门控通道活性，但它可能是细胞骨架系统的一部分，该系统用于维持视杆外段质膜内63 kDa通道复合物的组织。
