Nare B, Lubega G, Prichard R K, Georges E
Institute of Parasitology of McGill University, Macdonald Campus, Ste Anne de Bellevue, Quebec H9X 3V9, Canada.
J Biol Chem. 1996 Apr 12;271(15):8575-81. doi: 10.1074/jbc.271.15.8575.
Benzimidazoles (BZ) are broad spectrum anthelmintics thought to exert their effects by interacting with and disrupting the functions of microtubules. However, direct biochemical evidence for binding between BZ and tubulin has not been shown nor is it known what sequences in tubulin interact with BZ. In this study, a photoactive analogue of 2-acetamido-5-(3-aminophenoxy)benzimidaz ole that has biological activity similar to other benzimidazoles was synthesized and used to photoaffinity label cell lysates from the parasitic nematode of sheep Haemonchus contortus. The photoactive analogue, 2-acetamido-5-[3-(4-azido-3-125I-salicyl amido)phenoxy]benzimida zol e or 125I-ASA-BZ, was shown to photolabel a 54-kDa protein that was specifically immunoprecipitated with anti-tubulin monoclonal antibodies. Tubulin photoaffinity labeling by 125I-ASA-BZ was also inhibited with molar excess of various BZ analogues and colchicine. Interestingly, 125I-ASA-BZ photoaffinity-labeled the beta- and not the alpha-subunits of tubulin. Proteolytic digestion of 125I-ASA-BZ-labeled tubulin with Staphylococcus aureus V8 proteinase revealed one major peptide with an apparent molecular mass of 3.5 kDa. Exhaustive digestion of 125I-ASA-BZ-labeled beta-tubulin with trypsin resulted in two fractions containing radioactive peptides. Protein sequencing of the high performance liquid chromatography-purified tryptic ASA-BZ-photolabeled peptides identified the N-terminal 63-77 and 78-103 sequences as the BZ binding domain.
苯并咪唑（BZ）是一类广谱驱虫药，被认为通过与微管相互作用并破坏其功能来发挥作用。然而，尚未有BZ与微管蛋白结合的直接生化证据，也不清楚微管蛋白中哪些序列与BZ相互作用。在本研究中，合成了一种具有与其他苯并咪唑类似生物活性的2-乙酰氨基-5-（3-氨基苯氧基）苯并咪唑的光活性类似物，并用于对绵羊寄生线虫捻转血矛线虫的细胞裂解物进行光亲和标记。该光活性类似物，即2-乙酰氨基-5-[3-（4-叠氮基-3-¹²⁵I-水杨酰胺基）苯氧基]苯并咪唑或¹²⁵I-ASA-BZ，被证明可光标记一种54 kDa的蛋白质，该蛋白质能被抗微管蛋白单克隆抗体特异性免疫沉淀。¹²⁵I-ASA-BZ对微管蛋白的光亲和标记也受到各种BZ类似物和秋水仙碱的摩尔过量抑制。有趣的是，¹²⁵I-ASA-BZ光亲和标记的是微管蛋白的β亚基而非α亚基。用金黄色葡萄球菌V8蛋白酶对¹²⁵I-ASA-BZ标记的微管蛋白进行蛋白酶消化，得到一个表观分子量为3.5 kDa的主要肽段。用胰蛋白酶对¹²⁵I-ASA-BZ标记的β微管蛋白进行彻底消化，得到两个含有放射性肽段的组分。对高效液相色谱纯化的胰蛋白酶ASA-BZ光标记肽段进行蛋白质测序，确定N端63 - 77和78 - 103序列为BZ结合结构域。
