引用

致死因子(LF)是炭疽致命毒素的酶成分，它能特异性地裂解mapk激酶蛋白。蛋白水解与毒素发病机制之间的分子机制尚不清楚，然而，LF被认为是治疗抑制剂的理想靶点。识别潜在的抑制药物先导依赖于一种敏感和快速的方法筛选大量的化合物。这类检测最有效的方法之一是使用内淬荧光底物。这些底物包含与合适的显色基团配对的荧光基团，因此在裂解前荧光被荧光共振能量转移(FRET)猝灭。当裂解发生时，荧光恢复，酶活性可以连续监测。用于LF的FRET衬底MAPKKide已经在LIST实验室中设计出来。Km值为4.7 m。这种底物非常适合用于评估IC(50)s和潜在抑制剂的大屏幕抑制剂的抑制剂形式。一些金属蛋白酶抑制剂，包括抗生素，抗肽素，已经使用MAPKKide进行了检测。用LF测定actionin的IC(50)为18 m。Dixon图显示非竞争性抑制。 Inhibitor specificity for LF was confirmed using an unrelated enzyme that also cleaved MAPKKide. One disadvantage associated with FRET substrates is that the fluorophores absorb in the range of aromatic compounds that are potential inhibitors, thus complicating the interpretation of the data. Another FRET substrate for LF, using a fluorophore excited at longer wavelengths, has been designed at LIST laboratories. This FRET substrate can also be detected using an argon ion laser.