Lubega G W, Prichard R K
Institute of Parasitology, McGill University, Ste-Anne de Bellevue, QC, Canada.
Biochem Pharmacol. 1991 Jan 1;41(1):93-101. doi: 10.1016/0006-2952(91)90015-w.
The low- and high-affinity binding of tritiated benzimidazole anthelmintics (mebendazole and oxibendazole) to tubulin-containing supernatants derived from unembryonated eggs, third stage larvae or adult worms of thiabendazole-susceptible and -resistant strains of Haemonchus contortus were examined and compared. The displacement of these radioligands by unlabelled benzimidazoles (mebendazole, fenbendazole, thiabendazole and oxibendazole) also was examined. The binding affinity, K alpha, and maximum binding, Bmax, for the high-affinity binding were calculated by non-linear least-square iterative curve fitting using a computer programme (LIGAND) based on the exact mathematical model of ligand-receptor interactions. The K alpha was of the same order of magnitude (x 10(7) M-1) for the susceptible and resistant eggs, larvae and worms. Resistance was associated with a loss of high-affinity binding. There was a 2- to 5-fold loss of Bmax by the resistant strain. The eggs showed greater high-affinity binding per milligram of protein than the larvae which, in turn, showed greater high-affinity binding than the adult worms. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis that the tubulin content per milligram of protein decreased from egg, through larvae to adult worm. Cross-displacement studies indicated that different benzimidazole drugs interacted with the same receptor (tubulin) and that a rank order of affinity of the benzimidazole drugs could be inferred.
研究并比较了氚标记的苯并咪唑驱虫药（甲苯咪唑和奥苯达唑）与来自未受精虫卵、三期幼虫或噻苯达唑敏感及耐药捻转血矛线虫成虫的含微管蛋白上清液的低亲和力和高亲和力结合情况。还研究了未标记的苯并咪唑（甲苯咪唑、芬苯达唑、噻苯达唑和奥苯达唑）对这些放射性配体的置换作用。基于配体-受体相互作用的精确数学模型，使用计算机程序（LIGAND）通过非线性最小二乘迭代曲线拟合计算高亲和力结合的结合亲和力Kα和最大结合量Bmax。敏感和耐药的虫卵、幼虫及成虫的Kα处于相同数量级（×10⁷ M⁻¹）。耐药与高亲和力结合的丧失有关。耐药菌株的Bmax损失了2至5倍。每毫克蛋白质中，虫卵显示出比幼虫更高的高亲和力结合，而幼虫又比成虫显示出更高的高亲和力结合。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹分析表明，每毫克蛋白质中的微管蛋白含量从虫卵到幼虫再到成虫逐渐降低。交叉置换研究表明，不同的苯并咪唑药物与同一受体（微管蛋白）相互作用，并且可以推断出苯并咪唑药物的亲和力顺序。
