Jaffe Charles L, Dwyer Dennis M
Cell Biology Section, Div. of Intramural Research, NIAID, NIH, Bethesda, MD 20892, USA.
Parasitol Res. 2003 Oct;91(3):229-37. doi: 10.1007/s00436-003-0960-0. Epub 2003 Aug 16.
Protease activity was found in spent culture medium collected from Leishmania donovani, L. mexicana, L. major, as well as the insect trypanosomatids, Crithidia luciliae and Leptomonas seymouri. Released protease activity increased linearly over time and was correlated to promastigote density. In SDS-PAGE, zymogram gels showed that the protease's molecular weight ranged from 43-100 kDa. Spent culture medium proteases were blocked by the metallo-protease inhibitors, 1,10-phenanthroline and Z-Tyr-Leu-NHOH, but not by bestatin, leupeptin, ABESF, pepstatin A, E-64 or aprotinin. Monoclonal and/or polyclonal antibodies to the leishmanial gp63 reacted with the released Crithidia, Leptomonas, L. major and L. donovani proteases. Cell surface biotinylation and immune precipitation using gp63-specific antibodies showed that >34% of the released protease originated from the surface. Antibodies against the Trypanosoma brucei variable surface glycoprotein cross-reactive determinant (CRD) did not recognize this activity, suggesting that the gp63 is not cleaved from the cell surface by a parasite phospholipase, but is released by an alternative mechanism.
在从杜氏利什曼原虫、墨西哥利什曼原虫、硕大利什曼原虫以及昆虫锥虫（如路氏锥虫和西氏利什曼原虫）收集的用过的培养基中发现了蛋白酶活性。释放的蛋白酶活性随时间呈线性增加，且与前鞭毛体密度相关。在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）中，酶谱凝胶显示该蛋白酶的分子量范围为43 - 100 kDa。用过的培养基中的蛋白酶被金属蛋白酶抑制剂1,10 - 菲咯啉和Z - Tyr - Leu - NHOH阻断，但不被抑氨肽酶、亮抑酶肽、ABESF、胃蛋白酶抑制剂A、E - 64或抑肽酶阻断。针对利什曼原虫gp63的单克隆和/或多克隆抗体与释放的路氏锥虫、利什曼原虫、硕大利什曼原虫和杜氏利什曼原虫蛋白酶发生反应。使用gp63特异性抗体进行细胞表面生物素化和免疫沉淀表明，>34%的释放蛋白酶源自细胞表面。针对布氏锥虫可变表面糖蛋白交叉反应决定簇（CRD）的抗体未识别这种活性，这表明gp63不是由寄生虫磷脂酶从细胞表面切割下来的，而是通过另一种机制释放的。
