Research of Toxoplasma specific enzyme (practical use for diagnosis)

用高效液相色谱法从弓形虫滋养体及其体外培养液中分离纯化出两种NTPase同工酶(I、II)。然后测定ATP和ADP的Michaelis-Menten动力学常数，比较两种同工酶之间的差异。NTPase-I和NTPase-II对ATP和ADP的Vmax比值分别为150和1.1。NTPase-I和NTPase-II对ATP和ADP的Km值之比分别为0.12和1。结果表明，NTPase-I优先于ATP而不是ADP，NTPase-II对两种底物的水解率几乎相同。所有被调查的弓形虫菌株都具有NTPase-II的特性。因此，推测NTPase-II是一种较好的血清学诊断抗原。然而，通过双向免疫扩散、中和和Western-blotting分析，我们不能确定NTPase-I和NTPase-II的抗原性差异。这些数据有力地表明，这两种同工酶在其分子中都有一个相同的表位，该表位是主要的抗原点。使用NTPase进行血清学诊断，不需要考虑取决于菌株的同工酶类型。由于抗NTPase抗体在弓形虫感染急性期特异性升高，推测NTPase主要以快速分裂的速殖子形式存在。通过比较速殖子和慢性感染缓殖子的NTPase含量，证实了这一点。关于NTPase在血清诊断中的实际应用，这些结果已经发表在1992年5月的《临床微生物学杂志》上。其他结果目前正在准备中。

Two NTPase isozymes (I,II) were purified from the trophozoites of Toxoplasma gonii (RH) and its in vitro culture fluid by using a high performance liquid chromatography. Then the Michaelis-Menten kinetic constants for ATP and ADP were determined to compare the difference between the isozymes. The Vmax value ration for ATP and ADP of NTPase-I was 150, and that of NTPase-II was 1.1 , respectively. The Km value ratio for ATP and ADP of NTPase-I was 0.12, and that of NTPase-II was 1, respectively. The results indicated that NTPase-I preferred ATP to ADP and NTPase-II hydrolyzed both substrates at almost same rate. The properties of NTPase-II were present to all Toxoplasma strains as surveyed. Therefore, it is supposed that NTPase-II is a good antigen for serodiagnosis. However, we could not identify the antigenic difference between NTPase-I and NTPase-II by double-immunodiffusion, neutralization and western-blotting analysis. The data strongly suggest that both isozymes have a same epitope in their molecules and the epitope is a main antigenic site. Using NTPase for serodiagnosis, it is not necessary to consider the isozyme type which depends on the strain. Because anti-NTPase antibody increased specifically at acute phase infection of Toxoplasma, it was supposed that NTPase should be present mainly to fast-dividing tachyzoite form. This was confirmed by comparing the NTPase contents between tachyzoite and chronically infected bradyzoite. For practical use of NTPase to serodiagnosis, the results were already published by Journal Clinical Microbiology, May, 1992. Other results are now under preparation.