Haase W, Friese W, Gordon R D, Müller H, Cook N J
Max-Planck-Institut für Biophysik, Abteilung für Physiologie, Frankfurt/Main, Federal Republic of Germany.
J Neurosci. 1990 May;10(5):1486-94. doi: 10.1523/JNEUROSCI.10-05-01486.1990.
The sodium/calcium exchanger was purified from bovine retinal rod outer segment membranes and used for the immunization of New Zealand White rabbits. A polyclonal antibody was produced which was found to bind specifically to the 230 kDa Na+/Ca2(+)-exchanger protein as assessed by Western blotting. The antibody did not bind to the high-molecular-weight "rim protein," thereby demonstrating that this protein is distinct from the rod outer segment of Na+/Ca2(+)-exchanger. We used the polyclonal antibody for immunohistochemically localizing the exchange protein in bovine retina. Fluorescent light microscopy revealed intensive immunolabeling of the photoreceptor outer segments, whereas other retinal cell layers exhibited minimal binding. Using the electron microscopic immunogold method, we found specific antibody binding to the extracellular side of rod outer segment plasma membrane. Rod disk membranes, rod inner segments, and cone photoreceptors displayed no significant labeling. We therefore conclude that the Na+/Ca2(+)-exchanger is localized primarily in the rod outer segment plasma membrane, the most appropriate localization considering its proposed role in the process of vertebrate phototransduction.
从牛视网膜杆状细胞外段膜中纯化出钠/钙交换蛋白，并用于免疫新西兰白兔。产生了一种多克隆抗体，通过蛋白质印迹法评估发现该抗体能特异性结合230 kDa的Na+/Ca2(+)-交换蛋白。该抗体不与高分子量的“边缘蛋白”结合，从而证明这种蛋白与Na+/Ca2(+)-交换蛋白的杆状细胞外段不同。我们使用该多克隆抗体对牛视网膜中的交换蛋白进行免疫组织化学定位。荧光显微镜显示光感受器外段有强烈的免疫标记，而其他视网膜细胞层的结合最少。使用电子显微镜免疫金法，我们发现特异性抗体结合在杆状细胞外段质膜的细胞外侧。杆状盘膜、杆状细胞内段和锥体细胞光感受器没有明显的标记。因此，我们得出结论，Na+/Ca2(+)-交换蛋白主要定位于杆状细胞外段质膜，考虑到其在脊椎动物光转导过程中的假定作用，这是最合适的定位。
