Possani L D, Martin B M, Yatani A, Mochca-Morales J, Zamudio F Z, Gurrola G B, Brown A M
Departamento de Bioquímica, Instituto de Biotecnología, Universidad Nacional Autónoma de Mécixo, Cuernavaca-Morelos.
Toxicon. 1992 Nov;30(11):1343-64. doi: 10.1016/0041-0101(92)90511-3.
Taicatoxin is a new complex oligomeric toxin that was isolated from the venom of the Australian taipan snake Oxyuranus scutellatus scutellatus. It is composed of three different molecular entities: an alpha-neurotoxin-like peptide of mol. wt 8000, a neurotoxic phospholipase of mol. wt of 16,000 and a serine protease inhibitor of mol. wt 7000, linked by non-covalent bonds, at an approximate stoichiometry of 1:1:4. The most active form of the complex was isolated by ion exchange chromatography through DE-Cellulose followed by two steps of CM-Cellulose chromatography at pH 4.7 and pH 6.0, respectively. At this stage the complex migrates as a single component in beta-alanine-acetate-urea gel electrophoresis and is very toxic to mice (1 or 2 micrograms of the complex protein kills a mouse of 20 g within 2 hr). It blocks the high threshold calcium channel current of excitable membranes in heart and does not affect the low threshold calcium channel current. The block occurs at a site that is accessible extracellularly but not intracellularly. The block is selective for calcium channels, reversible, does not affect single channel conductance but only changes channel gating, and is voltage dependent with higher affinity for inactivated channels. The phospholipase activity of the complex toxin can be separated by affinity-chromatography using a phospholipid analog (PC-Sepharose). The resulting complex contains only alpha-neurotoxin and protease inhibitor and is still capable of blocking calcium channels, although with less potency than the native oligomeric form. Sephadex G-50 gel filtration chromatography in the presence of high salt (1M NaCl) at alkaline pH (8.2), separates the alpha-neurotoxin-like peptide from the protease inhibitor, but at this stage the resulting peptides lose physiological activity towards the calcium channels. The amino acid sequence of the protease inhibitor was determined by automatic Edman degradation. The alpha-neurotoxin-like peptide and two isosubunits displaying phospholipase activity were sequenced at the N-terminal part of the molecule.
太攀蛇毒素是一种新的复合寡聚毒素，它是从澳大利亚太攀蛇（Oxyuranus scutellatus scutellatus）的毒液中分离出来的。它由三种不同的分子实体组成：分子量为8000的α-神经毒素样肽、分子量为16000的神经毒性磷脂酶和分子量为7000的丝氨酸蛋白酶抑制剂，它们通过非共价键连接，化学计量比约为1:1:4。通过DE-纤维素离子交换色谱法，随后分别在pH 4.7和pH 6.0条件下进行两步CM-纤维素色谱法，分离出该复合物的最活跃形式。在此阶段，该复合物在β-丙氨酸-醋酸盐-尿素凝胶电泳中作为单一成分迁移，对小鼠具有很强的毒性（2小时内，1或2微克的复合蛋白可杀死一只20克重的小鼠）。它阻断心脏中可兴奋膜的高阈值钙通道电流，而不影响低阈值钙通道电流。这种阻断发生在细胞外可及但细胞内不可及的位点。这种阻断对钙通道具有选择性、可逆性，不影响单通道电导，仅改变通道门控，并且具有电压依赖性，对失活通道具有更高的亲和力。复合毒素的磷脂酶活性可以通过使用磷脂类似物（PC-琼脂糖）的亲和色谱法分离。所得复合物仅含有α-神经毒素和蛋白酶抑制剂，并且仍然能够阻断钙通道，尽管效力低于天然寡聚形式。在碱性pH（8.2）下，在高盐（1M NaCl）存在的情况下进行Sephadex G-50凝胶过滤色谱法，可将α-神经毒素样肽与蛋白酶抑制剂分离，但在此阶段，所得肽对钙通道失去生理活性。蛋白酶抑制剂的氨基酸序列通过自动埃德曼降解法测定。在分子的N端部分对α-神经毒素样肽和两个显示磷脂酶活性的异亚基进行了测序。
