el Aoumari A, Dupont E, Fromaget C, Jarry T, Briand J P, Kreitman B, Gros D
Laboratoire de Biologie de la Différenciation Cellulaire, UA CNRS 179, Faculté des Sciences de Luminy, Marseille/France.
Eur J Cell Biol. 1991 Dec;56(2):391-400.
Antipeptide antibodies directed to residues 55 to 66 (NTQQPGCENVCY) of connexin43 (cx43) specifically recognize this protein on Western blots of intact and urea-split gap junctions isolated from rat heart. These antibodies detect a single protein of 43 kDa, corresponding to cx43, on Western blots of whole fractions of various vertebrate hearts. Immunogold labeling by electron microscopy shows that the epitopes recognized by these antibodies are not localized on the cytoplasmic surfaces of intact gap junctions but only at the edges of these junctions. In urea-split gap junctions the gold particles are seen in the junctional space, associated with the extracellular faces of junctional membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat heart gap junctions treated with trypsin show that they are constituted with either two polypeptides of Mr 12,000 and 14,000 or a single polypeptide of Mr 22,000 according to whether the analyses are performed under reducing or non-reducing conditions, respectively. The antibodies directed to residues 55 to 66 of cx43 cross-react with both the 12 and 22 kDa polypeptides. These results suggest that the two protected domains of 12 and 14 kDa which contain the first extracellular loop and a putative second extracellular loop, respectively, are linked by disulfide bonds. In adult rat heart sections analyzed by indirect immunofluorescence the intercalated discs are labeled with antibodies directed to a cytoplasmic carboxy-terminal domain of cx43 (El Aoumari et al., J. Membr. Biol. 115, 229-240 (1990)). The same intercalated discs are also labeled in adjacent sections incubated with the antibodies directed to residues 55 to 66. Two hypotheses might explain these results: either the antibodies have access to the extracellular domain of cx43 molecules localized at the edges of the gap junctions, or cx43 molecules are present in the non-junctional membranes of the intercalated discs.
针对连接蛋白43（cx43）第55至66位氨基酸残基（NTQQPGCENVCY）的抗肽抗体，在从大鼠心脏分离的完整和经尿素裂解的缝隙连接的蛋白质免疫印迹中能特异性识别该蛋白。在各种脊椎动物心脏的全部分馏物的蛋白质免疫印迹中，这些抗体检测到一条43 kDa的单一蛋白条带，对应于cx43。通过电子显微镜进行的免疫金标记显示，这些抗体识别的表位并不位于完整缝隙连接的细胞质表面，而仅位于这些连接的边缘。在经尿素裂解的缝隙连接中，金颗粒出现在连接间隙中，与连接膜的细胞外表面相关联。对用胰蛋白酶处理的大鼠心脏缝隙连接进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，根据分析是在还原还是非还原条件下进行，它们分别由分子量为12,000和14,000的两种多肽或分子量为22,000的单一多肽组成。针对cx43第55至66位氨基酸残基的抗体与12 kDa和22 kDa的多肽都发生交叉反应。这些结果表明，分别包含第一个细胞外环和一个假定的第二个细胞外环的12 kDa和14 kDa的两个保护结构域通过二硫键相连。在通过间接免疫荧光分析的成年大鼠心脏切片中，闰盘被针对cx43细胞质羧基末端结构域的抗体标记（El Aoumari等人，《膜生物学杂志》115, 229 - 240 (1990)）。在用针对第55至66位氨基酸残基的抗体孵育的相邻切片中，相同的闰盘也被标记。有两种假设可以解释这些结果：要么抗体能够接触到位于缝隙连接边缘的cx43分子的细胞外结构域，要么cx43分子存在于闰盘的非连接膜中。
