Adamus G, Zam Z S, Arendt A, Palczewski K, McDowell J H, Hargrave P A
Department of Ophthalmology, University of Florida, Gainesville 32610.
Vision Res. 1991;31(1):17-31. doi: 10.1016/0042-6989(91)90069-h.
A panel of anti-bovine rhodopsin monoclonal antibodies (MAbs) of defined site-specificity has been prepared and used for functional and topographic studies of rhodopsins. In order to select these antibodies, hybridoma supernatants that contained anti-rhodopsin antibodies have been screened by enzyme-linked immunosorbent assay (ELISA) in the presence of synthetic peptides from rhodopsin's cytoplasmic regions. We selected for antibodies against predominantly linear determinants (as distinct from complex assembled determinants) and have isolated antibodies that recognize rhodopsin's amino terminus, its carboxyl terminus, as well as the hydrophilic helix-connecting regions 61-75, 96-115, 118-203, 230-252 and 310-321. Detailed specificities have been further determined by using a series of overlapping peptides and chemically modified rhodopsins as competitors. A group of seven antibodies with epitopes clustered within the amino terminal region of rhodopsin and a group of 15 antibodies with epitopes within the carboxyl terminal region are described. These MAbs have high affinities for rhodopsin with Kas in the range of 10(8)-10(10) M-1. Some MAbs specific for the carboxyl and amino terminal regions were used to compare these bovine rhodopsin sequences to those of different vertebrates. The MAbs cross-reacted with the different species tested to different extents indicating that there is some similarity in the sequences of these regions. However, some differences in the sequences were indicated by a reduced or absent cross-reactivity with some MAbs. In membrane topographic studies the MAbs showed both the presence and the accessibility of rhodopsin sequences 330-348, 310-321 and 230-252 on the cytoplasmic surface of the disk membrane. Similarly, sequences 1-20 and 188-203 were shown to reside on the lumenal surface of the disk and to be accessible to a macromolecular (antibody) probe. Antibodies directed against rhodopsin's carboxyl terminal sequence did not bind well to highly phosphorylated rhodopsin. Similarly, these antibodies as well as those against the V-VI loop inhibited phosphorylation of rhodopsin. Antibody A11-82P, specific for phosphorylated rhodopsin, recognized rhodopsin containing two or more phosphates and inhibited its further phosphorylation.
已制备了一组具有明确位点特异性的抗牛视紫红质单克隆抗体（MAb），并将其用于视紫红质的功能和拓扑学研究。为了筛选这些抗体，在视紫红质细胞质区域的合成肽存在下，通过酶联免疫吸附测定（ELISA）对含有抗视紫红质抗体的杂交瘤上清液进行了筛选。我们选择了针对主要线性决定簇（与复杂组装决定簇不同）的抗体，并分离出了识别视紫红质氨基末端、羧基末端以及亲水性螺旋连接区域61 - 75、96 - 115、118 - 203、230 - 252和310 - 321的抗体。通过使用一系列重叠肽和化学修饰的视紫红质作为竞争剂，进一步确定了详细的特异性。描述了一组七个抗体，其表位聚集在视紫红质的氨基末端区域内，以及一组15个抗体，其表位在羧基末端区域内。这些单克隆抗体对视紫红质具有高亲和力，解离常数（Ka）在10⁸ - 10¹⁰ M⁻¹范围内。一些针对羧基和氨基末端区域的单克隆抗体被用于比较这些牛视紫红质序列与不同脊椎动物的序列。这些单克隆抗体与所测试的不同物种有不同程度的交叉反应，表明这些区域的序列存在一些相似性。然而，与一些单克隆抗体的交叉反应降低或不存在表明序列存在一些差异。在膜拓扑学研究中，单克隆抗体显示视紫红质序列330 - 348、310 - 321和230 - 252在盘膜细胞质表面的存在和可及性。同样，序列1 - 20和188 - 203显示位于盘的腔表面，并可被大分子（抗体）探针所及。针对视紫红质羧基末端序列的抗体与高度磷酸化的视紫红质结合不佳。同样，这些抗体以及针对V - VI环的抗体抑制视紫红质的磷酸化。特异性针对磷酸化视紫红质的抗体A11 - 82P识别含有两个或更多磷酸基团的视紫红质，并抑制其进一步磷酸化。
