Alfieri S C, Pral E M, Shaw E, Ramazeilles C, Rabinovitch M
Departamento de Parasitologia, Universidade de São Paulo, Brazil.
Exp Parasitol. 1991 Nov;73(4):424-32. doi: 10.1016/0014-4894(91)90066-6.
Living Leishmania amazonensis amastigotes were incubated with radioiodinated N-benzyloxycarbonyl-L-tyrosyl-L-alanyl diazomethane (Z-Tyr-AlaCHN2), an irreversible inhibitor of mammalian cathepsins B and L. Parasite lysates were subjected to electrophoresis in gelatin-containing sodium dodecyl sulfate-acrylamide gels to detect regions of proteolytic activity, and the distribution of the inhibitor was ascertained by autoradiography. Of the three main bands of proteolysis associated with cysteine proteinases, two, with apparent molecular weights of 28 and 31 kDa, were shown to be labeled. The third enzyme activity, detected at the 35-kDa region in substrate gels, was only faintly labeled. The distribution of labeled bands was similar when lysates of untreated parasites were electrophoresed and the gels incubated with the radioiodinated inhibitor. Under reducing conditions, the inhibitor bound to polypeptides of 29, 31, 32, and 34 kDa, of which the first and the last were the most intensely labeled. Polypeptides with the same apparent molecular weights were labeled when amastigote lysates were incubated with the 125I inhibitor. Uptake of radioactivity by the parasites was time and concentration-dependent and more than 80% of the total counts could be precipitated with trichloroacetic acid. Radioactivity associated with the amastigotes was quite stable after they were pulsed with labeled inhibitor and chased for up to 24 hr in inhibitor-free medium. Both total uptake and labeling of cysteine proteinases were markedly reduced in parasites preincubated with Z-Phe-AlaCHN2 prior to exposure to Z-Tyr(125I)-AlaCHN2. However, more radioiodinated inhibitor was taken up by parasites preincubated with cold inhibitor and chased in inhibitor-free medium, suggesting de novo synthesis or processing of inactive enzyme precursors.
将活的亚马逊利什曼原虫无鞭毛体与放射性碘化的N-苄氧羰基-L-酪氨酰-L-丙氨酰重氮甲烷（Z-Tyr-AlaCHN2）一起孵育，Z-Tyr-AlaCHN2是一种哺乳动物组织蛋白酶B和L的不可逆抑制剂。将寄生虫裂解物在含明胶的十二烷基硫酸钠-丙烯酰胺凝胶中进行电泳，以检测蛋白水解活性区域，并通过放射自显影确定抑制剂的分布。在与半胱氨酸蛋白酶相关的三个主要蛋白水解条带中，有两个条带的表观分子量分别为28 kDa和31 kDa，显示有标记。在底物凝胶中35 kDa区域检测到的第三种酶活性仅被微弱标记。当未处理寄生虫的裂解物进行电泳，凝胶与放射性碘化抑制剂孵育时，标记条带的分布相似。在还原条件下，抑制剂与29 kDa、31 kDa、32 kDa和34 kDa的多肽结合，其中第一个和最后一个被标记得最强烈。当无鞭毛体裂解物与125I抑制剂孵育时，具有相同表观分子量的多肽被标记。寄生虫对放射性的摄取具有时间和浓度依赖性，总计数的80%以上可以用三氯乙酸沉淀。在用标记抑制剂脉冲并在无抑制剂培养基中追踪长达24小时后，与无鞭毛体相关的放射性相当稳定。在用Z-Tyr(125I)-AlaCHN2处理之前，先用Z-Phe-AlaCHN2预孵育的寄生虫中，半胱氨酸蛋白酶的总摄取和标记均显著降低。然而，先用冷抑制剂预孵育并在无抑制剂培养基中追踪的寄生虫摄取了更多的放射性碘化抑制剂，这表明无活性酶前体的从头合成或加工。
