Study for pyrimidine de novo biosynthetic pathway of Toxoplasma gondii(application for development of anti-protozoan drug)

本研究的目的是在项目期间寻找调节弓形虫嘧啶从头合成途径的主要因子。为了进行该项目的研究，通过检查测量化合物的分析方法来研究核酸前体、嘌呤和嘧啶核苷酸的细胞含量。用高效液相色谱法测定了弓形虫细胞内的核苷酸浓度，其灵敏度在实验范围内。另一方面，本研究尝试了嘧啶从头合成途径的第一步酶-氨甲酰磷酸合成酶-II（carboxamylphosphate synthetase-II，CPS-II）的基因克隆。在此之前，美国达特茅斯大学的一个研究小组已经报道了CPS-II基因的全长序列。 ...更多信息 无论如何，从我们的结果和达特茅斯大学的数据中可以清楚地确定，弓形虫CPS-II中不存在最有效的CPS-II激活剂PRPP结合位点。我们与达特茅斯大学的研究小组开展了一个合作项目，研究弓形虫CPS-II的酶调节，并试图制备全尺寸的重组CPS-II。遗憾的是，由于弓形虫CPS-II是一个巨大的蛋白质，重组CPS-II的表达尚未成功。虽然还没有进行酶学研究，CPS-II的全氨基酸序列的数据强烈表明，有助于从头嘧啶生物合成途径的流动的最重要的因素是ATP的供应，CPS-II的底物。由于宿主细胞的ATP污染，很难准确测定弓形虫细胞内ATP的浓度，但生长期速殖子细胞内ATP的浓度（~ 5 mM）明显高于休眠期速殖子细胞内ATP的浓度（~ 1 mM）。考虑到弓形虫CPS-II对ATP的km值（约10 mM），在休眠状态下可能几乎不存在酶活性。这些结果有力地表明，能量的供应是从头嘧啶生物合成流程中最重要的因素。该项目的目的几乎达到了，尽管许多事实需要进一步证实。少

The aim of this study is to find out a primary factor that regulates a flow of de novo pyrimidine biosynthetic pathway in Toxoplasma gondii within term of the project. To perform a study of the project, the cellular contents of nucleic acid precursors, purine and pyrimidine nucleotides, were investigated by examining an analytical method to measure the compounds. The nucleotide concentrations in T.gondii cell were measurable by a high performance liquid chromatography and the sensitivity of the measurement was within an experimental condition. For another approach to the project, a cloning of the gene encoding carbamylphosphate synthetase-II (CPS-II) that is the first step enzyme and the most important enzyme of pyrimidine de novo biosynthetic pathway, was attempted. Consequently, a partial cDNA clone encoding a substrate binding site of CPS-II was obtained, however, full length of CPS-II gene had been reported by a group of Dartmouth University in the United States before our success. … More Anyway, it was clearly identified that there is no PRPP, the most potent activator of CPS-II, binding site in the T.gondii CPS-II from our results and the data from the group of Dartmouth University. We started a collaboration project with the group of Dartmouth University to study an enzyme regulation of T.gondii CPS-II and tried to make a full size of recombinant CPS-II. Unfortunately, the expression of the recombinant CPS-II has not been successful because T.gondii CPS-II is a huge protein. Although an enzymological study has not been performed, the data of full amino acid sequence of CPS-II strongly suggests that the most important factor that contributes to the flow of de novo pyrimidine biosynthetic pathway is a supply of ATP, a substrate of CPS-II. An accurate assay of ATP concentration in T.gondii cell is very hard because of the ATP contamination from host cell, nevertheless, the estimated concentration of ATP in growing tachyzoite cell (〜5 mM) is quite higher than that of dormant cell (〜1 mM). Considering the km value of T.gondii CPS-II for ATP (〜10 mM), almost no enzyme activity is probably present under dormant condition. The results strongly suggest that the supply of energy is the most important factor for the flow of de novo pyrimidine biosynthesis. The aim of this project was almost achieved, though many facts should be further confirmed. Less