REGULATION OF METABOLISM IN PARASITIC HELMINTHS

寄生蠕虫代谢的调节

• 批准号: 3136890
• 负责人: Ben Gerald Harris
• 金额: $ 15.56万
• 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 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3136890
• 关键词: 6 phosphofructokinase; Ascaris; actins; affinity chromatography; analytical ultracentrifugation; antineoplastics; bioenergetics; biological information processing; carbohydrate metabolism; circular dichroism; cyclic AMP; drug design /synthesis /production; enzyme mechanism; enzyme structure; gel electrophoresis; gel filtration chromatography; glycogenolysis; glycolysis; immunoprecipitation; ion exchange chromatography; ligase; malate dehydrogenase; mitochondria; pentosyltransferase; phosphorylation; protein kinase; proteolysis; spectrometry; ultraviolet spectrometry

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 their 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 regulation of the very important glycolytic rate-limiting enzyme, phosphofructokinase (PFK) as well as identification and characterization of a phosphofructo-2-kinase (PFK-2). The study will also pursue the chemical mechanism of an important mitochondrial enzyme, NAD-malice enzyme. The studies on the ascarid PFK will the development of a physiological assay for the enzyme. This assay will be based on the levels of known effectors of the PFK and will be involved with correlating the activity of the enzyme in vitro that which can calculated to occur in vivo. The primary effectors studied will be AMP, fructose-2,6- bisphosphate and covalent phosphorylation of the PFK. The next set of studies will be on the mechanism and structure of the ascarid PFK. Studies on the mechanism of the enzyme will be conducted on the PFK that has been desensitized by derivatization by reaction of several histidine residues with diethylpyrocarbonate (d-PFK). They will involve studies on the kinetic mechanism of the enzyme using the techniques of isotope partitioning and positional isotope exchange. Regulatory mechanism studies will use the same effectors as those specified above. This study will measure the precise influence of the effectors on various rate processs of the enzyme. The chemical mechanism of the d-PFK will be probed with the use of pH and residue studies in order to identify groups involved in binding and catalysis. Structural studies will be conducted using amino acid sequencing of peptides form the residue studies above in order to identify areas of the PFK involved in the active and regulatory sites. Spectral studies will be conducted with UV- visible spectrophotometry, native tryptophan fluorescence and circular dichroism studies in order to probe the conformational changes taking place during binding of substrates and effectors. Tryptic studies will also be conducted to probe the overall structure of the enzyme. PFK-2 will be isolated and purified from the muscle of the ascarid. Physico-chemical studies will be carried out on the enzyme as well as studies of its ability to be stimulated by phosphorylation. PFK-2 will also be tested for fructose-2.6-bisphosphatase activity. Finally, kinetic studies on the NAd-malic enzyme will be carried out on the reverse reaction, the reductive carboxylation of pyruvate by CO2 and NADH.

该研究计划的总体目标是开发 清楚地了解碳水化合物和能量代谢 寄生蠕虫。 这一目标的本质是界定 控制碳流动的监管步骤 这些途径。这些监管步骤的完整描述 他们的调制器将提供关于 寄生虫及其与其环境的关系方式， 主持人。 有了这些信息，就可以设计 化疗药物的作用方式基于 寄生虫与其宿主之间的差异。 这些研究将 对寄生线虫、猪蛔虫进行 专注于非常重要的糖酵解的调节 限速酶、磷酸果糖激酶 (PFK) 以及 磷酸果糖-2-激酶的鉴定和表征 （PFK-2）。 该研究还将探究其化学机制 重要的线粒体酶，NAD-恶意酶。 有关的研究 蛔虫 PFK 将开发一种生理测定法 酶。 该测定将基于已知的水平 PFK 的效应器并将参与关联 可以计算出的酶的体外活性 体内。 研究的主要效应器是 AMP、果糖-2,6- PFK 的二磷酸和共价磷酸化。 下一组 研究将集中在蛔虫的机制和结构上 PFK。 对该酶的作用机制进行研究 通过反应衍生化脱敏的PFK 用焦碳酸二乙酯 (d-PFK) 处理几个组氨酸残基。 他们将涉及酶的动力学机制的研究 使用同位素分配和定位同位素技术 交换。 调节机制研究将使用相同的效应器 如上所述。 这项研究将精确测量 效应器对酶的各种速率过程的影响。 d-PFK 的化学机制将通过使用 pH 值和残留物研究，以确定参与的群体 结合和催化。 结构研究将使用 对上述残留物研究中的肽进行氨基酸测序 为了确定 PFK 参与活动和活动的领域 监管站点。 光谱研究将使用 UV- 可见分光光度法、天然色氨酸荧光法和 圆二色性研究以探测构象 底物和效应物结合期间发生的变化。 还将进行胰蛋白酶研究以探讨整体 酶的结构。 PFK-2将从中分离和纯化 蛔虫的肌肉。 物理化学研究将 对这种酶进行了研究，并研究了它的能力 受磷酸化刺激。 PFK-2也将进行测试 果糖-2.6-二磷酸酶活性。 最后，动力学研究 NAd-苹​​果酸酶会进行逆反应， CO2 和 NADH 使丙酮酸还原羧化。