Lockwood B C, North M J, Scott K I, Bremner A F, Coombs G H
Mol Biochem Parasitol. 1987 May;24(1):89-95. doi: 10.1016/0166-6851(87)90119-8.
A highly sensitive electrophoretic method involving gelatin-containing polyacrylamide gels has been used to analyse trichomonad proteinases. Multiple forms, optimally active at pH 5-6, were present in all four species examined, but the species could be distinguished from one another by both quantitative and qualitative differences. The intestinal parasites, Trichomitus batrachorum and Pentatrichomonas hominis, had lower specific activities than the urogenital parasites, Trichomonas vaginalis and Tritrichomonas foetus, and, in the case of P. hominis, there were fewer enzyme forms. The high activity proteinases of Tritrichomonas foetus had low apparent molecular weights (less than 25 kDa), while the predominant enzymes of Trichomonas vaginalis were of high apparent molecular weight (68-110 kDa). Distinct differences were also observed between the proteinase patterns of various isolates of T. vaginalis. All of the enzymes were stimulated by dithiothreitol, suggesting that they were cysteine proteinases. This was confirmed for the T. vaginalis and Tritrichomonas foetus proteinases from their inhibition by antipain, leupeptin, TLCK and iodoacetic acid. The method allows the detection of proteinases in samples of Trichomonas vaginalis containing as few as 10(4) cells or as little as 1 microgram protein. It was also possible to detect proteinase activity released into the medium. For both T. vaginalis and Tritrichomonas foetus, the extracellular enzymes present during early log phase were qualitatively different from the intracellular proteinases, although the latter were present in samples of media obtained from later cultures (cell densities greater than 1 X 10(5) parasites ml-1). The results show the potential of this technique for detecting proteinases in trichomonad samples in studies aimed at determining proteinase function in pathogenesis and host-parasite relationships.
一种涉及含明胶聚丙烯酰胺凝胶的高灵敏度电泳方法已被用于分析毛滴虫蛋白酶。在所检测的所有四个物种中均存在多种在pH 5 - 6时活性最佳的形式，但这些物种可通过定量和定性差异相互区分。肠道寄生虫巴氏三毛滴虫和人五毛滴虫的比活性低于泌尿生殖道寄生虫阴道毛滴虫和胎儿三毛滴虫，而且就人五毛滴虫而言，酶的形式较少。胎儿三毛滴虫的高活性蛋白酶表观分子量较低（小于25 kDa），而阴道毛滴虫的主要酶表观分子量较高（68 - 110 kDa）。在阴道毛滴虫的各种分离株的蛋白酶谱之间也观察到明显差异。所有这些酶都受到二硫苏糖醇的刺激，表明它们是半胱氨酸蛋白酶。阴道毛滴虫和胎儿三毛滴虫的蛋白酶被抗蛋白酶、亮抑酶肽、甲苯磺酰-L-赖氨酸氯甲基酮和碘乙酸抑制，从而证实了这一点。该方法能够检测阴道毛滴虫样品中低至10⁴个细胞或低至1微克蛋白质中的蛋白酶。也能够检测释放到培养基中的蛋白酶活性。对于阴道毛滴虫和胎儿三毛滴虫，对数早期阶段存在的细胞外酶在性质上与细胞内蛋白酶不同，尽管后者存在于后期培养物（细胞密度大于1×10⁵个寄生虫/毫升）获得的培养基样品中。结果表明，在旨在确定蛋白酶在发病机制和宿主 - 寄生虫关系中的功能的研究中，该技术在检测毛滴虫样品中的蛋白酶方面具有潜力。
