PURINE METABOLISM IN SCHISTOSOMA MANSONI

曼索尼血吸虫的嘌呤代谢

• 批准号: 3565495
• 负责人: Ching Chung WANG
• 金额: $ 16.81万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-08-01 至 1992-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3565495
• 关键词: Escherichia coli; Schistosoma mansoni; X ray crystallography; anthelmintics; complementary DNA; computer graphics /printing; drug design /synthesis /production; enzyme inhibitors; enzyme mechanism; enzyme structure; gene expression; genetic library; genetic manipulation; genetic mapping; hypoxanthine phosphoribosyltransferase; laboratory mouse; molecular cloning; nucleic acid sequence; parasitic disease chemotherapy; peptide chemical synthesis; plasmids; protein sequence; purine /pyrimidine metabolism; restriction mapping; schistosomiasis; tubercidin

One of the potential benefits of comparative biochemical studies of parasites is the identification of metabolic deficiencies in the latter and the exploitation of these vulnerabilities for antiparasitic chemotherapy. We have recently verified the absence of de novo synthesis of purine nucleotides in Schistosoma mansoni and proceeded to delineate the purine salvage pathways in this organism. Our results pointed to hypoxanthine-guanine phosphoribosyl transferase (HGPRT) as one of the pivotal enzymes in the purine salvage by S. mansoni; an effective inhibition of this enzyme would lead to death of the parasite. We have since purified this enzyme to apparent homogeneity, partially characterized it and found many of its properties differ from those of the mammalian HGPRT; thus raising the possibility of selective inhibition of the parasite HGPRT. Our future research plan calls for; (1) searching for specific and potent inhibitors of S. mansoni HGPRT and pursuing their possible use as antischistosomal agents; (2) cloning the c-DNA encoding S. mansoni HGPRT and comparing its restriction map and nucleotide sequences with the known amino acid sequence of mammalian HGPRT for possible differences in protein structures between the two enzymes; (3) cloning the full-length c-DNA of S. mansoni HGPRT. This will be done either by introducing a specially constructed expression vector plasmid into an Escherichia coli #165 strain deleted in xanthine-guanine phosphoribosyl transferase (XGPRT) for expression; or by introducing a c-DNA library of S. mansoni constructed in retroviral LTR chimeric plasmids into a mammalian HGPRT cell line and selecting for the expression of S. mansoni HGPRT in HAT medium. The native S. mansoni HGPRT thus produced in larger quantities by these methods will facilitate more thorough investigations on the kinetic and structural of the enzyme; (4) cloning the genomic DNA encoding S. mansoni HGPRT and comparing its full size, restriction pattern and nucleotide sequences with the full-length S. mansoni HGPRT c-DNA to search for possible introns in the gene structure and any regulatory segments flanking the gene in order to better understand the genetic regulation of S. mansoni HGPRT. We have also identified a tubercidin kinase in S. mansoni which has a unique substrate specificity and is the target of the potent antischistosomal activity of tubercidin. We plan to further characterize this enzyme as a potential target for antischistosomal chemotherapy.

比较生物化学研究的一个潜在好处是， 寄生虫是后者代谢缺陷的鉴定， 利用这些弱点进行抗寄生虫化疗。 我们最近证实了没有从头合成嘌呤 曼氏血吸虫的核苷酸，并继续描绘嘌呤 在这个有机体中的补救途径。 我们的研究结果表明， 次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HGPRT）作为一种 关键酶的嘌呤补救的S。有效的 这种酶的抑制将导致寄生虫的死亡。 我们有 由于将该酶纯化至表观均一性， 发现它的许多特性与哺乳动物的不同 从而提高了选择性抑制寄生虫的可能性 HGPRT。 我们未来的研究计划要求：（1）寻找特定的， S. mansoni HGPRT，并寻求其可能的用途， （2）克隆编码S.曼索尼HGPRT 并将其限制性酶切图谱和核苷酸序列与已知的 哺乳动物HGPRT的氨基酸序列，用于蛋白质中可能的差异 （3）克隆了S. mansoni HGPRT。 这将通过引入一个专门的 将构建的表达载体质粒导入大肠杆菌#165菌株 黄嘌呤-鸟嘌呤磷酸核糖转移酶（XGPRT）缺失， 表达;或通过引入S.曼索尼建于 将逆转录病毒LTR嵌合质粒导入哺乳动物HGPRT细胞系， 选择表达S. mansoni HGPRT在HAT培养基中培养。 天然 S.因此，通过这些方法大量生产的mansoni HGPRT将 促进对动力学和结构的更彻底的调查， （4）克隆编码S. mansoni HGPRT和 将其全长、限制性酶切图谱和核苷酸序列与 全长S. mansoni HGPRT c-DNA来寻找可能的内含子， 基因结构和基因侧翼的任何调控片段， 为了更好地了解S. mansoni HGPRT。 我们有 还在S. mansoni有一个独特的 底物特异性，是强效抗组胺药的靶点。 杀结核菌素的活性。 我们计划进一步将这种酶定性为 抗血吸虫化疗的潜在靶点。