Garcia M P, Nóbrega O T, Teixeira A R, Sousa M V, Santana J M
Departamento de Biologia Celular, Universidade de Brasília, Brasília-DF, Brazil.
Mol Biochem Parasitol. 1998 Mar 15;91(2):263-72. doi: 10.1016/s0166-6851(97)00205-3.
A novel proteolytic activity was identified in epimastigote, amastigote and trypomastigote forms of Trypanosoma cruzi using the fluorogenic substrate N-Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Epimastigotes showed enzyme activity to be 2-fold higher than amastigotes and trypomastigotes. The protease that displays this activity was purified from epimastigote forms by a four step chromatographic procedure: Diethylaminoethyl-Sephacel, Phenyl-Sepharose, Phenyl-Superose, and Concanavalin A Sepharose columns. The purified enzyme is a glycoprotein that migrates as a 30 kDa protein in 12.5% SDS-polyacrylamide gel electrophoresis (PAGE), under reducing conditions. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. The inhibition pattern of the purified 30 kDa protease showed that it belongs to the cysteine-protease class. In addition to the synthetic substrate, the purified protease hydrolysed bovine serum albumin (BSA) and human type I collagen. The N-terminal amino acid sequence of the protease shows similarity to the mammalian cathepsin B protease.
使用荧光底物N-琥珀酰-亮氨酰-亮氨酰-缬氨酰-酪氨酸-7-氨基-4-甲基香豆素，在克氏锥虫的上鞭毛体、无鞭毛体和锥鞭毛体形式中鉴定出一种新型蛋白水解活性。上鞭毛体的酶活性比无鞭毛体和锥鞭毛体高2倍。通过四步色谱法从鞭毛体形式中纯化出具有这种活性的蛋白酶：二乙氨基乙基-琼脂糖凝胶、苯基-琼脂糖凝胶、苯基-超级凝胶和伴刀豆球蛋白A琼脂糖凝胶柱。纯化后的酶是一种糖蛋白，在还原条件下，在12.5%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（PAGE）中以30 kDa的蛋白形式迁移。发现其对荧光底物和蛋白质底物的最佳酶活性均出现在酸性pH值下。纯化的30 kDa蛋白酶的抑制模式表明它属于半胱氨酸蛋白酶类。除了合成底物外，纯化后的蛋白酶还能水解牛血清白蛋白（BSA）和人I型胶原蛋白。该蛋白酶的N端氨基酸序列与哺乳动物组织蛋白酶B蛋白酶相似。
