THE TOXOPLASMA GONDII NUCLEOTIDE TRIPHOSPHATE HYDROLASE AND NUCLEOSIDE SALVAGE

弓形虫是一种专性细胞内寄生虫， 在宿主细胞内的一种独特的膜包围的空泡。像所有 胞内寄生原虫T.弓形虫必须从 宿主细胞为了生存和复制 寄生虫周围的宿主细胞内是自由渗透到小 分子，为寄生虫提供现成的mM浓度 从宿主细胞胞质中提取ATP。最常见的蛋白质之一 由寄生虫产生的（总寄生虫蛋白的1-2%）是核苷 三磷酸水解酶（NTH），其依次切割所有核苷 将三磷酸盐转化为二磷酸盐和非磷酸盐形式。新台币 是由寄生虫分泌的，对于ATP酶来说，这是一个非常不寻常的过程， 并积聚在液泡空间和液泡膜上。在 在这个位点，酶可以通过提供 AMP与膜结合寄生虫5 '-核苷酸酶结合，随后 产生高水平的腺苷。三个随机重复的基因组 已经鉴定了NTR的拷贝，其中两个在 RH强毒株，在核苷酸上的相同性超过97%， 氨基酸水平同工型的ATP酶活性显著高于对照组， 不同，只有活性较低的同种型在两个非- 毒性，生长缓慢的分离株，提高了NTT的可能性， 表达与细胞内的复制潜能有关。 NTT作为一种潜在的治疗药物将被深入研究 作为液泡空间和膜中靶点的范例 接受新的干预策略。实验提出了 为了进一步表征NTR同工型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同种型， 研究表达的调节和阶段特异性，并寻找 其他球虫寄生虫的同源物。 的结构和功能 将通过确定亚基组成、亚基组成和亚基组成来研究NTPase。 酶动力学和各种亚型的亚细胞分布， 通过描绘参与核苷酸结合和二巯基的残基， 介导的活性调节，通过鉴定功能性 活性，并通过评估酶在核苷补救中的作用， 和其他功能。

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.