Hawrot E, Wilson P T, Gershoni J M, Reese J H, Lentz T L
Brain Res. 1986 May 14;373(1-2):227-34. doi: 10.1016/0006-8993(86)90335-5.
The binding of [125I]iodo-alpha-bungarotoxin [( 125]alpha-BuTX) to the dissociated alpha-subunit of Torpedo acetylcholine receptor (AChR) can be readily demonstrated in a modified 'protein-blot' analysis utilizing electrophoretically transferred, dissociated subunits immobilized onto positively charged nylon membranes which are then incubated directly with [125I]alpha-BuTX. We report here the use of the protein-blotting technique to detect the alpha-BuTX binding site present in the central nervous system of lower vertebrates and to characterize some of the physicochemical properties of the toxin binding site. High molecular weight (Mr greater than or equal to 200,000 and greater than or equal to 120,000) alpha-BuTX-binding components can be readily demonstrated in avian and fish brain extracts upon protein-blotting with [125I]alpha-BuTX following lithium dodecyl sulfate PAGE. Neither extensive reduction with dithiothreitol nor prior reduction followed by alkylation with iodoacetamide alter the mobility of the CNS-derived BuTX-binding sites. In contrast to our findings with Torpedo AChR or muscle AChR derived from a number of different species, no binding is observed in the molecular weight range of the alpha-subunit (Mr = 40,000) nor is any binding at any molecular weight observed in similar fractions prepared from adult, mammalian (rat, guinea pig) brain using this technique. These results demonstrate the existence in lower vertebrate brain of a BuTX binding site comparable in size to the AChR oligomeric complex of electric organ and muscle. They also suggest, however, striking structural differences between muscle AChR and the central neuronal BuTX-binding complex as well as a considerable difference between the neuronal BuTX-binding sites derived from lower and higher vertebrate brain.
在一种改良的“蛋白质印迹”分析中，利用电泳转移并固定在带正电荷尼龙膜上的解离亚基，然后直接用[125I]碘-α-银环蛇毒素（[125I]α-BuTX）孵育，可轻易证明[125I]碘-α-银环蛇毒素（[125]α-BuTX）与电鳐乙酰胆碱受体（AChR）的解离α亚基的结合。我们在此报告使用蛋白质印迹技术来检测低等脊椎动物中枢神经系统中存在的α-BuTX结合位点，并表征毒素结合位点的一些物理化学性质。在用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）后用[125I]α-BuTX进行蛋白质印迹时，在鸟类和鱼类脑提取物中可轻易证明高分子量（Mr≥200,000和≥120,000）的α-BuTX结合成分。用二硫苏糖醇进行广泛还原或先用二硫苏糖醇还原后再用碘乙酰胺烷基化，均不会改变中枢神经系统来源的BuTX结合位点的迁移率。与我们对来自许多不同物种的电鳐AChR或肌肉AChR的研究结果相反，在α亚基的分子量范围内（Mr = 40,000）未观察到结合，并且使用该技术从成年哺乳动物（大鼠、豚鼠）脑制备的类似组分中，在任何分子量下均未观察到结合。这些结果证明在低等脊椎动物脑中存在一个大小与电器官和肌肉的AChR寡聚复合物相当的BuTX结合位点。然而，它们也表明肌肉AChR与中枢神经元BuTX结合复合物之间存在显著的结构差异，以及低等和高等脊椎动物脑来源的神经元BuTX结合位点之间存在相当大的差异。
