THE TOXOPLASMA GONDII NUCLEOTIDE TRIPHOSPHATE HYDROLASE AND NUCLEOSIDE SALVAGE

弓形虫是一种专性的细胞内寄生虫，它居住在 宿主细胞内有一种独特的膜结合液泡。像所有人一样 细胞内的原生动物寄生虫弓形虫必须从 宿主细胞为了生存和复制液泡膜 寄主细胞内寄生虫的周围可以自由渗透到小的 分子，为寄生虫提供了随时获取mm浓度的途径 从宿主细胞胞浆中提取的ATP。最普遍的蛋白质之一 由寄生虫(占寄生虫总蛋白的1-2%)产生的是一种核苷 顺序裂解所有核苷的三磷酸水解酶(NTPase) 三磷酸盐以二磷酸盐和非磷酸盐形式存在。NTPase 是由寄生虫分泌的，这对ATPase来说是一个明显不寻常的过程， 并聚集在液泡空间和液泡膜上。在… 在这个位置，该酶可以通过提供 AMP结合到膜结合的寄生虫5‘-核苷酸酶，随后 产生高水平的腺苷。三个随机重复的基因组 已经确定了NTPase的拷贝，其中两个拷贝在 与强毒RH株的核苷酸同源性超过97%。 氨基酸水平。这些异构体的ATPase活性惊人地 不同的且只有活性较低的异构体在两个非 毒力强、生长缓慢的分离物，增加了NTPase 其表达与细胞内的复制潜能有关。 NTPase将作为一种潜在的治疗方法进行深入研究 靶标和作为空泡空间和膜中靶标的范例 服从于新的干预策略。提出了实验方案。 进一步研究NTPase异构体在不同组织中的表达 寄生虫菌株，通过遗传手段删除和补充NTP亚型，以 研究表达的调节和阶段特异性，并寻找 其他球虫寄生虫中的同源物。的结构和功能 将通过确定亚基组成来研究NTPase， 酶动力学和各种异构体的亚细胞分布， 通过描述参与核苷酸结合的残基和二硫醇- 通过识别功能抑制物介导的活性调节 活性，并通过评估该酶在核苷回收中的作用 以及液泡空间内的其他功能。

Toxoplasma gondii is an obligate intracellular parasite which resides in a unique membrane bounded vacuole inside host cells. Like all intracellular protozoan parasites, T. gondii must salvage purines from the host Cell in order to survive and replicate The vacuolar membrane surrounding the parasite inside host cells is freely permeable to small molecules, providing the parasite with ready access to mM concentrations of ATP from the host Cell cytosol. One of the most prevalent proteins produced by the parasite (1-2% of total parasite protein) is a nucleoside triphosphate hydrolase (NTPase) which sequentially cleaves all nucleoside triphosphates to the di-phosphate and the non-phosphate form. The NTPase is secreted by the parasite, a distinctly unusual process for an ATPase, and accumulates in the vacuolar space and at the vacuolar membrane. At this site, the enzyme could participate in purine salvage by supplying AMP to a membrane bound parasite 5'-nucleotidase, with subsequent generation of high levels of adenosine. Three tandomly repeated genomic copies of the NTPase have been identified, two of which are expressed in the virulent RH strain, and are over 97% identical at the nucleotide and amino acid level. The ATPase activity of the isoforms is strikingly different and only the less active isoform is expressed in two non- virulent, slow-growing isolates, raising the possibility that NTPase expression is related to replicative potential inside cells. The NTPase will be investigated in depth as a potential therapeutic target and as a paradigm for targets in the vacuolar space and membrane amenable to novel strategies for intervention. Experiments are proposed to further characterize expression of NTPase isoforms in different parasite strains, to delete and replete NTP isoforms by genetic means, to study the regulation and stage specificity of expression, and to look for homologues in other coccidian parasites. The structure and function of the NTPase will be studied by determining the subunit composition, the enzyme kinetics and the subcellular distribution of the various isoforms, by delineating residues involved in nucleotide binding and dithiol- mediated regulation of activity, by identifying inhibitors of functional activity, and by evaluating the role of the enzyme in nucleoside salvage and other functions within the vacuolar space.