REGULATION OF METABOLISM IN PARASITIC HELMINTHS

寄生蠕虫代谢的调节

• 批准号: 2003390
• 负责人: Ben Gerald Harris
• 金额: $ 19.3万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2003390
• 关键词: 6 phosphofructokinase; Ascaris; bioenergetics; carbohydrate metabolism; circular dichroism; enzyme mechanism; enzyme structure; enzyme substrate; glycolysis; high performance liquid chromatography; host organism interaction; nucleic acid sequence; phosphorylation; protein kinase; protein structure function; proteolysis; radionuclides

The overall objective of this research program is to develop a clear understanding of carbohydrate and energy metabolism in parasitic helminths. Inherent in this objective is the delineation of the regulatory steps which control the flow of carbon through these pathways. A complete description of these regulatory steps and the modulators would provide unique information on the parasite and the manner in which it relates to its environment, the host. With this information, it might be possible to design chemotherapeutic agents whose mode of action would be based on the differences between the parasite and its host. These studies will be carried out on the parasitic nematode, Ascaris suum, and will concentrate on the enzyme, phosphofructokinase (PFK). A cDNA clone has been isolated that presumably contains the full sequence of PFK. It will be sequenced and then expressed in a bacterial vector. The PFK will be used to study the residues which participate in catalysis and regulation. They will be studied by derivatization with various reagents that specifically react with that residue. Carboxyls will be derivatized with N-ethyl-5- phenylisoxazolium-3'sulfonate (Woodward Reagent K), cysteines will be modified with N-ethylmaleiimide, lysines in the ATP inhibitory site with 2',3'-dialdehyde ATP histidines with diethylpyrocarbonate, and tyrosines with tetranitromethane and N-acetylimidazole. Derivatization will be by radioactive reagents and protection from enzyme inactivation by substrates and effectors will be noted. Then the enzyme will be digested with a protease and the radioactive peptides will be isolated by HPLC and sequenced with a gas-phase sequenator. With the knowledge of the sequence obtained earlier, we will be able define those residues that participate in catalysis and regulation and thus define portions of the molecule that contain the active and regulatory sites. We will also work on the kinetic mechanism of the enzyme by studying isotope partitioning and positional isotope exchange which will give information on the rate limiting steps of mechanism. We will also work on pH studies which will define those groups that must be protonated or unprotonated for catalysis or regulation. By doing these studies combined with those chemical derivatization studies above, important residues will be distinguished. Using this information plus knowledge of other PFKs, we will mutagenize certain residues, express and isolate the mutant protein and study the effect of the mutant amino acid on catalysis or regulation. In this manner, we should be able to predict what the catalytic mechanism should be. We will also study the structure of the PFK as it relates to function. The conditions will be determined under which the tetramer dissociates into the dimers or monomers. The circular dichroic spectra of the PFK will be determined when it is in its native state versus that when it is phosphorylated. We will also note the differences in the spectra when d-PFK and pd-PFK are run. Finally, utilizing the recombinant PFK a crystallization study will be begun in which the various forms of the PFK that we have developed (n-PFK, d-PFK, pn-PFK, pd PFK, o-PFK) will be tested for their ability to crystallize in the presence substrates, products and effectors.

这项研究计划的总体目标是制定一项明确的 对寄生生物碳水化合物和能量代谢的认识 蠕虫。这一目标所固有的是对监管的界定 通过这些途径控制碳流动的步骤。一个完整的 这些监管步骤和调节器的描述将提供 关于寄生虫的唯一信息及其与之相关的方式 它的环境，宿主。有了这些信息，就有可能 设计其作用模式将基于 寄生虫和它的宿主之间的差异。这些研究将是 对寄生线虫猪蛔虫进行了研究，并将集中 在酶，磷酸果糖激酶(PFK)。已分离到一个cdna克隆。 这可能包含了PFK的全序列。它将被排序 然后在细菌载体中表达。PFK将被用于研究 参与催化和调节的残基。他们将会是 通过使用各种特定反应的试剂进行衍生化研究 带着这些残留物。羧基将被N-乙基-5-酮衍生。 苯基异恶唑-3‘磺酸盐(伍德沃德试剂K)，半胱氨酸 用N-乙基马来酰亚胺修饰ATP抑制部位的赖氨酸 2‘，3’-二醛-三磷酸腺苷、二乙基焦碳酸酯和酪氨酸 四硝基甲烷和N-乙酰咪唑。衍生化将由 放射性试剂与底物对酶失活的防护 效应器将被注意到。然后这种酶将用一种 蛋白水解酶和放射性多肽将通过高效液相色谱仪和 用气相测序仪测序。在知道序列的情况下 ，我们将能够确定那些参与的残基 在催化和调节中，从而定义了分子中 包含活动站点和监管站点。我们还将致力于研究 从同位素分配和定位研究该酶的作用机理 同位素交换，将提供有关速率限制步骤的信息 机制。我们还将进行PH值研究，以确定这些群体 必须质子化或去质子化才能催化或调节。通过 将这些研究与那些化学衍生化研究相结合 在上面，重要的残基将被区分开来。使用此信息 加上其他PFK的知识，我们将突变某些残留物，表示 并分离突变蛋白，研究突变氨基酸的作用。 对酸的催化或调节作用。通过这种方式，我们应该能够 预测催化机理应该是什么。我们亦会研究 与功能相关的PFK的结构。条件将是 确定四聚体在哪个条件下解离成二聚体或 单体。将在以下条件下测定PFK的圆二色光谱 它处于天然状态，而不是被磷酸化时。我们会 还要注意运行d-pfk和pd-pfk时光谱的差异。 最后，利用重组PFK进行了结晶研究。 开始其中我们已经开发的各种形式的PFK(n-PFK， D-pfk、pn-pfk、pd-pfk、o-pfk)将被测试其能力 在存在的底物、产品和效应器中结晶。