Kuwahara T, Paulin J J, Cosgrove W B, Agosin M
Department of Zoology, University of Georgia, Athens 30602.
Arch Biochem Biophys. 1988 Jul;264(1):214-21. doi: 10.1016/0003-9861(88)90587-5.
The cytosolic flavin enzyme from Trypanosoma cruzi was isolated by a modification of the previously reported method (T. Kuwahara, R. A. White, Jr., and M. Agosin (1985) Arch. Biochem. Biophys. 239, 18-28). In the present study, rabbits were inoculated with the purified enzyme and antibodies were purified from the sera. Ouchterlony double-diffusion analysis indicated that the antibodies reacted specifically with the flavoenzyme and not with other T. cruzi proteins. At the equivalence point, 1 ml of antibody neutralized about 4 nmol of enzyme. The IgG fraction had a small inhibitory effect on the catalytic activity of the enzyme as measured by cytochrome c reduction but only at IgG concentrations well above the equivalence point. Immunotitration of the enzyme in T. cruzi cultures showed that the enzyme corresponds to about 1% of the total protein during the logarithmic phase of growth, but this value decreases to about 0.6% during the stationary phase. Among various trypanosomatids tested, T. cruzi had the highest enzyme concentration; whereas, in other species it ranged from 0.25 to 2.4 micrograms/mg protein. These marked differences suggest that the antibody may be suitable for taxonomic purposes. The presence of the enzyme in amastigotes maintained in tissue culture cells was demonstrated by indirect immunofluorescence. The enzyme was found localized in the periphery of the cell, just beneath the subpellicular microtubules. However, distribution of the enzyme in epimastigotes was more diffuse. As immunofluorescence could be detected only in amastigotes and not in the tissue culture cells, it is suggested that the antibody may be suitable for histopathological diagnosis of Chagas' disease.
克氏锥虫的胞质黄素酶是通过对先前报道的方法（T. 桑原、R. A. 怀特、小怀特和M. 阿戈辛（1985年）《生物化学与生物物理学报》239卷，第18 - 28页）进行改进而分离得到的。在本研究中，用纯化后的酶对兔子进行接种，并从血清中纯化抗体。欧氏双扩散分析表明，该抗体与黄素酶特异性反应，而不与其他克氏锥虫蛋白反应。在等价点时，1毫升抗体可中和约4纳摩尔的酶。IgG组分对以细胞色素c还原法测定的酶催化活性有轻微抑制作用，但仅在IgG浓度远高于等价点时才有此作用。对克氏锥虫培养物中的酶进行免疫滴定表明，在对数生长期，该酶约占总蛋白的1%，但在稳定期，这一数值降至约0.6%。在测试的各种锥虫中，克氏锥虫的酶浓度最高；而在其他物种中，其范围为0.25至2.4微克/毫克蛋白。这些显著差异表明该抗体可能适用于分类学目的。通过间接免疫荧光法证明了在组织培养细胞中维持的无鞭毛体中存在该酶。发现该酶定位于细胞周边，恰好在表膜下微管下方。然而，该酶在无鞭毛体中的分布更为弥散。由于仅在无鞭毛体中可检测到免疫荧光，而在组织培养细胞中未检测到，因此表明该抗体可能适用于恰加斯病的组织病理学诊断。
