Tomalski M D, Kutney R, Bruce W A, Brown M R, Blum M S, Travis J
Department of Entomology, University of Georgia, Athens 30602.
Toxicon. 1989;27(10):1151-67. doi: 10.1016/0041-0101(89)90009-3.
Three low mol. wt proteins which have contracting-paralyzing activity in insects were isolated from extracts of the straw itch mite, Pyemotes tritici. One of these toxins, referred to as TxP-I, was purified to apparent homogeneity using the following sequence: ion-exchange, affinity, hydroxyapatite and reverse-phase chromatography. The other two toxins, referred to as TxP-II, remained as a mixture. Peptide mapping and immunoblot analysis suggest that TxP-I and TxP-II are probably isoproteins. The apparent mol. wt of native TxP-I and of the two components of TxP-II were 27,000, 28,000 and 29,000, respectively. The apparent mol. wt of the toxins after reductive carboxamidomethylation increased to 38,000, 41,000 and 43,000, respectively. The amino acid composition of TxP-I indicates a high content of Cys (8 mole%). Therefore, several disulfide bonds may impart a very compact tertiary structure to this protein which, upon denaturation, unfolds and increases its Stoke's radius resulting in retarded mobility on a polyacrylamide gel. The N-terminal sequence of TxP-I is not homologous with any other protein for which the sequence is known. The paralysis dose50 of TxP-I (PD50) in wax moth larvae is ca. 500 micrograms/kg and it is not toxic to mice at a dose of 50 mg/kg. A polyclonal antibody, raised against TxP-I, reacted with both TxP-I and TxP-II. The antibody neutralized the rapid, muscle-contracting paralysis of these toxins. Using this antibody and immunocytochemistry, we found the toxins localized in posterior glands which appear to be connected with the stylet through a series of ducts. We conclude that TxP-I and TxP-II are part of a complex mixture of neurotoxins which P. tritici utilizes to capture prey.
从麦扁螨（Pyemotes tritici）提取物中分离出三种在昆虫体内具有收缩 - 麻痹活性的低分子量蛋白质。其中一种毒素，称为TxP - I，通过以下步骤纯化至表观均一性：离子交换、亲和、羟基磷灰石和反相色谱法。另外两种毒素，称为TxP - II，仍为混合物。肽图谱和免疫印迹分析表明TxP - I和TxP - II可能是同型蛋白。天然TxP - I以及TxP - II的两种组分的表观分子量分别为27,000、28,000和29,000。还原羧甲基化后毒素的表观分子量分别增加到38,000、41,000和43,000。TxP - I的氨基酸组成表明其半胱氨酸含量很高（8摩尔％）。因此，几个二硫键可能赋予该蛋白质非常紧密的三级结构，变性后该结构展开并增加其斯托克斯半径，导致在聚丙烯酰胺凝胶上迁移受阻。TxP - I的N端序列与任何已知序列的其他蛋白质都不同源。TxP - I在蜡螟幼虫中的麻痹剂量50（PD50）约为500微克/千克，在50毫克/千克的剂量下对小鼠无毒。针对TxP - I产生的多克隆抗体与TxP - I和TxP - II都发生反应。该抗体中和了这些毒素引起的快速肌肉收缩麻痹。使用这种抗体和免疫细胞化学方法，我们发现毒素定位于后腺，后腺似乎通过一系列导管与口针相连。我们得出结论，TxP - I和TxP - II是麦扁螨用于捕获猎物的神经毒素复杂混合物的一部分。
