Research of Toxoplasma specific enzyme (practical use for diagnosis)

采用高效液相色谱法从gonoplasma gonii （RH）的滋养体及其体外培养液中纯化了2个NTPase同工酶（I，II）。然后测定ATP和ADP的Michaelis-Menten动力学常数，比较两种同工酶的差异。NTPase-I对ATP和ADP的Vmax比值为150，NTPase-II的Vmax比值为1.1。NTPase-I和NTPase-II对ATP和ADP的Km值比值分别为0.12和1。结果表明，相对于ADP， NTPase-I更倾向于ATP，而NTPase-II对两种底物的水解速率几乎相同。所有弓形虫株均具有NTPase-II的特性。因此，认为NTPase-II是一种良好的血清诊断抗原。然而，我们无法通过双免疫扩散、中和和western-blotting分析确定NTPase-I和NTPase-II的抗原差异。这两种同工酶在其分子中具有相同的表位，并且该表位是主要的抗原位点。使用NTPase进行血清诊断，不需要考虑同工酶的类型，这取决于菌株。由于抗NTPase抗体在弓形虫感染急性期特异性升高，推测NTPase主要存在于快速分裂的速殖子中。通过比较速殖子和慢性感染慢殖子的NTPase含量，证实了这一点。对于NTPase在血清诊断中的实际应用，研究结果已经发表在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.