DIFFERENTIAL IMMUNOGOLD DEXTRAN LABELING OF BOVINE AND FROG ROD AND CONE CELLS USING MONOCLONAL-ANTIBODIES AGAINST BOVINE RHODOPSIN
DIFFERENTIAL IMMUNOGOLD DEXTRAN LABELING OF BOVINE AND FROG ROD AND CONE CELLS USING MONOCLONAL-ANTIBODIES AGAINST BOVINE RHODOPSIN
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DOI：
10.1016/0014-4835(86)90017-5
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发表时间：
1986-01-01
影响因子：
3.4
通讯作者：
MOLDAY, RS
中科院分区：
文献类型：
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作者：
HICKS, D;MOLDAY, RS
采用11种抗牛视紫红质不同片段的单克隆抗体与免疫金-葡聚糖标记物对牛和蛙视网膜感光细胞的低丙基薄片进行标记，以便在透射电镜下观察。针对视紫红质c端、F1-F2环和n端的抗体均能密集标记牛棒外段（ROS），而对棒内段（RIS）仅能稀疏标记。大多数抗体每亩结合20-600个金颗粒。在ROS中，每亩10-60个金颗粒。每亩5-20个颗粒。m2在低辛基树脂上。一个针对n端和一个针对c端的抗体导致每亩超过1000个粒子的结合。m2在牛ROS中。锥外段（COS）仅用一种抗体rho 3D6标记，该抗体对牛视紫红质1“-4”c端具有特异性。90%的COS被观察到有这种抗体标记。免疫金葡聚糖标记法研究了这些抗体对红绿蛙ROS和COS中视紫红质的交叉反应性。单克隆抗体靶向F1-F2环上的位点，n端密集标记红色青蛙ROS，但不标记绿色ROS或COS。针对1”-8”段结合位点的三种c端特异性抗体分别标记绿色和红色ROS，但在绿色ROS上观察到更高程度的标记。抗体rho 3D6，发现牛COS，也标记青蛙COS。这些结果表明，牛视紫质的F1-F2环和沿n端和c端的片段与青蛙视紫质的相应区域具有高度的同源性；牛视紫质的c -末端1“-8”段与来自绿色活性氧的蛙视紫质的相应片段密切相关；牛视紫红质的1′-4′c端与大多数牛和蛙COS的视蛋白片段高度同源。讨论了sds -凝胶电泳法观察到的蛙红色活性氧与多种形式视紫红质的关系。
Eleven monoclonal antibodies against different segments of bovine rhodopsin were used with immunogold-dextran markers to label Lowicryl thin sections of bovine and frog retinal photoreceptor cells for visualization by transmission electron microscopy. Antibodies against the C-terminus, F1-F2 loop and N-terminus of rhodopsin were all observed to label bovine rod outer segments (ROS) densely, but to label rod inner segments (RIS) only sparsely. Most antibodies bound 20-600 gold particles per .mu.m2 in the ROS, 10-60 gold particles per .mu.m2 in the RIS and 5-20 particles per .mu.m2 on the Lowicryl resin. One antibody against the N-terminus and one antibody against the C-terminus resulted in the binding of over 1000 particles per .mu.m2 in bovine ROS. Cone outer segments (COS) were labeled with only one antibody, rho 3D6, having a specificity for 1''-4'' C-terminus of bovine rhodopsin. Ninety per cent of the COS were observed to be labeled with this antibody. Immunogold-dexran labeling was also used to study the cross-reactivity of these antibodies to rhodopsin in red and green frog ROS and COS. Monoclonal antibodies directed against sites along the F1-F2 loop, and the N-terminus labeled red frog ROS densely, but did not label either green ROS or COS. Three C-terminal specific antibodies against binding sites along the 1''-8'' segment labeled both green and red ROS, but a higher extent of labeling was observed on the green ROS. Antibody rho 3D6, which found to bovine COS, also labeled frog COS. These results indicate that the F1-F2 loop and segments along to the N-terminus and the C-terminus of bovine rhodopsin show a high degree of homology with corresponding regions of frog rhodopsin from red ROS; the C-terminal 1''-8'' segment of bovine rhodopsin is closely related to the corresponding segment of frog rhodopsin from green ROS; and the 1''-4'' C-terminus of bovine rhodopsin is highly homologous to segments of opsin from most bovine and frog COS. The labeling of frog red ROS in relation to multiple forms of rhodopsin observed by SDS-gel electrophoresis is discussed.