REGULATION OF METABOLISM IN PARASITIC HELMINTHS

这项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晰的认识 寄生蠕虫。这一目标所固有的是对 控制碳的流动的监管步骤 这些小路。对这些监管步骤的完整描述 它们的调制器将提供关于 寄生虫及其与环境相关的方式， 主持人。有了这些信息，就有可能设计出 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 在寄生线虫猪蛔虫上进行，并将 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-激酶的鉴定和性质 (PFK-2)。该研究还将探讨An的化学机制。 重要的线粒体酶，NAD-恶意酶。关于中国农业发展的研究 蛔虫PFK将建立一种生理学检测方法 这种酶。这项化验将基于已知的 PFK的效应器，并将参与关联 酶的体外活性可以计算出发生的情况 在活体内。研究的主要效应物将是AMP，果糖-2，6- PFK的二磷酸和共价磷酸化。下一盘 将对蛔虫的机制和结构进行研究 PFK。将对该酶的作用机理进行研究 通过反应衍生化而钝化的全氟辛烷基酮 几种组氨酸残基与焦碳酸二乙酯(d-PFK)的结合。 他们将涉及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 交换。监管机制研究将使用相同的效应器 如上所述。这项研究将测量准确的 效应器对酶的不同速率过程的影响。 对d-pfk的化学机理进行了探讨。 PH值和残留物研究，以确定参与的基团 结合和催化。结构研究将采用以下方式进行 来自上述残基研究的多肽的氨基酸序列 为了确定PFK涉及的活跃区域和 监管网站。光谱研究将使用UV- 可见光分光光度，天然色氨酸荧光和 用圆二色谱研究构象 在底物和效应器结合过程中发生的变化。 还将进行胰酶研究，以探索整体 酶的结构。PFK-2将从 蛔虫的肌肉。物理化学研究将是 对该酶进行了研究，并研究了其被 受磷酸化的刺激。还将对PFK-2进行测试 果糖-2.6-二磷酸酶活性。最后，进行了动力学研究 苹果酸酶将在逆反应中进行， CO2和NADH对丙酮酸的还原羧化作用。

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.