Research of Toxoplasma specific enzyme (practical use for diagnosis)

用高效液相色谱法从刚氏弓形虫（RH）滋养体及其体外培养液中分离纯化了两种NTR同工酶（Ⅰ、Ⅱ）。然后测定ATP和ADP的米氏动力学常数，比较同工酶之间的差异。NTPase-I对ATP和ADP的Vmax比值为150，NTPase-II为1.1。NTPase-I对ATP和ADP的Km值比为0.12，NTPase-II对ATP和ADP的Km值比为1。结果表明，NTPase-I对ATP的水解优先于ADP，NTPase-II对ATP和ADP的水解速率几乎相同。NTPase-II的特性存在于所调查的所有弓形虫株中。因此，NTPase-II是一种良好的血清学诊断抗原。然而，我们不能确定NTPase-I和NTPase-II之间的抗原性差异，通过免疫双扩散，中和和western-blotting分析。这些数据强烈地表明，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NTR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NTR抗体在弓形虫感染急性期特异性升高，推测NTR抗体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的缓殖子之间的NTR含量证实了这一点。关于NTR在血清学诊断中的实际应用，结果已在1992年5月的《临床微生物学杂志》上发表。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.