de Carvalho Thaís Batista, David Erica Boarato, Coradi Silvana Torossian, Guimarães Semíramis
Departamento de Parasitologia, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, São Paulo, 18618-000, Brazil.
Parasitol Res. 2008 Dec;104(1):185-90. doi: 10.1007/s00436-008-1185-z. Epub 2008 Sep 17.
There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in pathogenesis of giardiasis. This report describes a preliminary characterization of the proteolytic activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). The protease activity of E/S products in conditioned medium by trophozoites of each strain was analyzed using substrate (gelatin and collagen) impregnated SDS-PAGE and hemoglobin assay. The protease characterization was based on inhibition assays including synthetic inhibitors. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted degradation of the substrates and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibition assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of hemoglobin.
有证据表明，贾第虫滋养体含有和/或释放蛋白水解酶，这些酶可能与贾第虫病的发病机制有关。本报告描述了巴西无菌株（BTU-11）和参考株波特兰1（P1）的十二指肠贾第虫滋养体排泄/分泌（E/S）产物中蛋白水解活性的初步特征。使用底物（明胶和胶原蛋白）浸渍的SDS-PAGE和血红蛋白测定法分析了各菌株滋养体在条件培养基中E/S产物的蛋白酶活性。蛋白酶的表征基于包括合成抑制剂在内的抑制试验。在两种受试菌株的滋养体的条件培养基中均检测到蛋白水解产物。在含有共聚明胶和胶原蛋白的凝胶中，E/S产物促进了底物的降解，最明显的蛋白水解区分别分布在明胶水解和胶原蛋白水解活性模式下77至18 kDa和145至18 kDa的迁移区域。还观察到血红蛋白的降解，并且在两种受试E/S产物中水解模式相似。抑制试验表明，两种E/S产物中的主要蛋白水解活性归因于半胱氨酸蛋白酶，尽管也表明存在丝氨酸蛋白酶，主要在血红蛋白的水解中。
