ULTRASTRUCTURAL LOCALIZATION OF RHODOPSIN IN THE VERTEBRATE RETINA
ULTRASTRUCTURAL LOCALIZATION OF RHODOPSIN IN THE VERTEBRATE RETINA
复制标题
脊椎动物视网膜中视紫红质的超微结构定位
作者：
L. Jan;J. Revel
杜威和合作者的早期工作显示了青蛙视网膜中视紫红质的分布。我们使用单独针对视紫红质的抗体在牛和小鼠的眼睛上重复了这些实验。 emulphogene 中的牛视紫红质在羟基磷灰石柱上纯化。该试剂的纯度通过分光光度标准、十二烷基硫酸钠 (SDS) 凝胶电泳和等电聚焦确定。该视紫红质用作免疫吸附剂，从用溶解在 2% 洋地黄皂苷中的牛杆外节免疫的兔子的抗血清中分离出特异性抗体。免疫电泳显示如此制备的抗体属于IgG类并且不与牛杆外节的脂质提取物发生交叉反应。木瓜蛋白酶消化的单价抗体 (Fab) 与过氧化物酶结合用于标记甲醛固定的牛和鼠视网膜中的视紫红质。除了圆盘膜之外，还标记了外节的质膜、连接纤毛和部分杆内节膜。我们观察到杆外节质膜和盘膜两侧都有染色。免疫标记实验的结果与冷冻裂解样本中观察到的膜颗粒的观察结果之间存在差异。我们的实验表明，冷冻裂解实验中膜颗粒的分布反映了膜蛋白的分布。另一方面，除非极其小心地控制，否则免疫标记可能会引入几种不同类型的伪影。
Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.