Millership Jason J, Chappell Cynthia, Okhuysen Pablo C, Snowden Karen F
Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station 77843-4467, USA.
J Parasitol. 2002 Oct;88(5):843-8. doi: 10.1645/0022-3395(2002)088[0843:COAAFT]2.0.CO;2.
Microsporidia are obligate intracellular parasites of the phylum Microspora. To date, more than 1,200 species within 144 genera have been described, with 14 infecting humans. Currently, no effective treatment exists for human microsporidiosis. In this study, the biochemical properties of the aminopeptidases were investigated within several species of microsporidia. Aminopeptidase activity was detected in 3 species of microsporidia, Encephalitozoon cuniculi, E. hellem, and Vittaforma corneae, using a fluorometric substrate assay. Each species exhibited distinct aminopeptidase properties. The cytosolic neutral aminopeptidase activities of the Encephalitozoon spp. were characterized as preferentially cleaving leucine, whereas those of V. corneae cleaved arginine. Native polyacrylamide gel electrophoresis estimated the molecular mass of E. cuniculi, E. hellem, and V. corneae as 74, 72, and 79 kDa, respectively. Enzymatic activity was inhibited by bestatin and it's analogue, nitrobestatin, indicating that the enzyme was an aminopeptidase for all species. Inhibition with the chelating agents ethylenediaminetetraacetic acid and 1,10phenanthroline characterized the enzymes as metalloaminopeptidases. Subcellular fractionation of the 3 microsporidial species suggested that the enzyme activity was localized in the cytosolic fraction. Optimal enzyme activity was observed at pH 7.2 for all species. This is the first report of enzyme characterization from these 3 species of microsporidia.
微孢子虫是微孢子虫门的专性细胞内寄生虫。迄今为止，已描述了144个属中的1200多种，其中14种可感染人类。目前，人类微孢子虫病尚无有效的治疗方法。在本研究中，对几种微孢子虫的氨肽酶的生化特性进行了研究。使用荧光底物分析法在3种微孢子虫，即兔脑炎微孢子虫、海伦脑炎微孢子虫和角膜维塔微孢子虫中检测到了氨肽酶活性。每个物种都表现出独特的氨肽酶特性。脑炎微孢子虫属的胞质中性氨肽酶活性的特征是优先切割亮氨酸，而角膜维塔微孢子虫的则切割精氨酸。天然聚丙烯酰胺凝胶电泳估计兔脑炎微孢子虫、海伦脑炎微孢子虫和角膜维塔微孢子虫的分子量分别为74、72和79 kDa。酶活性被抑氨肽酶素及其类似物硝基抑氨肽酶抑制，表明该酶对所有物种都是氨肽酶。用螯合剂乙二胺四乙酸和1,10-菲咯啉抑制表明这些酶是金属氨肽酶。对这3种微孢子虫进行亚细胞分级分离表明酶活性定位于胞质部分。所有物种在pH 7.2时观察到最佳酶活性。这是关于这3种微孢子虫酶特性的首次报道。
