Nakayama H, Nakayama K, Nonomura Y, Kobayashi M, Kangawa K, Matsuo H, Kanaoka Y
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Biochim Biophys Acta. 1993 Jan 18;1145(1):134-40. doi: 10.1016/0005-2736(93)90390-l.
Three peptides corresponding to residues (13-23C), (Y1419-1431), and (1809-1820) of the electric eel sodium channel have been synthesized and used to raise antisera in rabbits. All the antibodies produced specifically recognized the corresponding peptides in an ELISA assay. However, their avidities to the channel protein were different. Two antibodies, against the sequence (1809-1820) and (13-23C) which are directed to the C-terminus region and to the adjacent portion of N-terminus, respectively, recognized the 250 kDa channel protein in the immunoblot and an ELISA assay. Binding of the two antibodies to the sodium channel in oriented electroplax membrane vesicles was increased 4-5-fold after permeabilizing the vesicles with 0.01% saponin, implying cytoplasmic orientation for the two peptides. The cytoplasmic orientation of the N- and C-terminal regions were further confirmed by immunogold electron microscopy. By contrast, antibody raised against the sequence (Y1419-1431) which has been proposed to be the transmembrane segment S4 of internal repeat IV, did not react with the channel protein not only after the saponin treatment but also even under the immunoblot conditions following SDS-PAGE. The antibody could recognize fragments of the channel protein after digestion with lysyl endoproteinase, suggesting that the region may apparently form a strictly well-ordered conformation in the transmembrane part of the channel molecule.
已合成了与电鳗钠通道的残基（13 - 23C）、（Y1419 - 1431）和（1809 - 1820）相对应的三种肽，并用于在兔体内产生抗血清。在酶联免疫吸附测定（ELISA）中，所产生的所有抗体都能特异性识别相应的肽。然而，它们对通道蛋白的亲和力不同。两种分别针对C末端区域和N末端相邻部分的序列（1809 - 1820）和（13 - 23C）的抗体，在免疫印迹和ELISA测定中识别出250 kDa的通道蛋白。在用0.01%皂角苷使膜泡通透后，这两种抗体与定向电鳐膜泡中钠通道的结合增加了4 - 5倍，这意味着这两种肽位于细胞质侧。通过免疫金电子显微镜进一步证实了N末端和C末端区域的细胞质定位。相比之下，针对已被认为是内部重复序列IV的跨膜片段S4的序列（Y1419 - 1431）产生的抗体，不仅在皂角苷处理后，而且在SDS - PAGE后的免疫印迹条件下都不与通道蛋白反应。该抗体在用赖氨酰内肽酶消化后能识别通道蛋白的片段，这表明该区域在通道分子的跨膜部分可能明显形成一种严格有序的构象。
