REGULATION OF METABOLISM IN PARASITIC HELMINTHS

寄生蠕虫代谢的调节

• 批准号: 3136888
• 负责人: Ben Gerald Harris
• 金额: $ 14.64万
• 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/3136888
• 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一种 对碳水化合物和能量代谢有清楚的了解， 寄生蠕虫 这一目标的本质是划定 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 它们的调制器将提供关于 寄生虫及其与环境的关系， 主持人 有了这些信息，也许可以设计出 化疗剂，其作用方式将基于 寄生虫和宿主之间的差异。 这些研究将 在寄生线虫猪蛔虫上进行， 专注于调节非常重要的糖酵解 限速酶磷酸果糖激酶（PFK）以及 一种磷酸果糖-2-激酶鉴定和性质研究 （PFK-2）。 这项研究还将探讨 重要的线粒体酶，NAD-恶意酶。 的研究 蛔虫PFK将发展一种生理测定方法， 酶 该测定将基于已知的 PFK的效应物，并将参与相关的 酶的体外活性，可计算发生 in vivo. 研究的主要效应物将是AMP、果糖-2，6-二磷酸腺苷（果糖-2，6-二磷酸腺苷）和果糖-2，6-二磷酸腺苷（果糖-2，6-二磷酸腺苷）。 二磷酸和PFK的共价磷酸化。 下一组 蛔虫的作用机制和结构将是研究的重点 PFK。 酶的作用机理研究将在 已通过反应衍生化脱敏的PFK 几个组氨酸残基与焦碳酸二乙酯（d-PFK）。 它们将涉及对酶的动力学机制的研究 利用同位素分配和位置同位素技术 交易所 调节机制研究将使用相同的效应物 如上文所述。 这项研究将测量精确的 效应子对酶各速率过程的影响。 本文还将用分子动力学方法探讨d-PFK的化学机理。 pH值和残留物研究，以确定参与 结合和催化。 结构研究将使用 从上述残基研究中对肽进行氨基酸测序 为了确定PFK的活动区域， 监管场所。 光谱研究将采用UV- 可见分光光度法，天然色氨酸荧光和 圆二色谱研究，以探测构象 在底物和效应物结合过程中发生的变化。 还将进行胰蛋白酶研究，以探索总体 酶的结构。 PFK-2将被分离和纯化， 蛔虫的肌肉 物理化学研究将 对酶进行了研究，并研究了其被 由磷酸化作用刺激。 PFK-2还将进行测试， 果糖-2.6-二磷酸酶活性。 最后， 将NAD-苹果酸酶进行上逆反应， 丙酮酸被CO2和NADH还原羧化。