REGULATION OF METABOLISM IN PARASITIC HELMINTHS

该研究计划的总体目标是制定一个明确的 了解寄生虫的碳水化合物和能量代谢 蠕虫这一目标的本质是划定监管机构的范围， 控制碳通过这些途径的步骤。一个完整 这些调节步骤和调节剂的描述将提供 关于寄生虫的独特信息及其与 它的环境，宿主。有了这些信息， 设计其作用模式将基于 寄生虫和宿主之间的差异。这些研究报告将 对寄生线虫猪蛔虫进行了研究， 磷酸果糖激酶（PFK）。已分离出一个cDNA克隆， 可能包含PFK的完整序列它将被测序 然后在细菌载体中表达。PFK将用于研究 参与催化和调节的残基。他们将 通过用各种试剂进行衍生化来研究， 与这些残留物。羧基将用N-乙基-5-甲基-N-乙基-N-甲基-N-乙基-N-甲基-N 苯基异恶唑钥-3 '磺酸盐（Woodward试剂K），半胱氨酸将被 用N-乙基马来酰亚胺修饰，ATP抑制位点的赖氨酸， 2 '，3'-二醛ATP组氨酸与焦碳酸二乙酯和酪氨酸 与四硝基甲烷和N-乙酰基咪唑反应。衍生化将通过 放射性试剂和防止底物使酶失活 并且将记录效应器。然后酶会被一种 蛋白酶和放射性肽将通过HPLC分离， 用气相测序仪测序。知道了这个序列 我们将能够定义那些参与 在催化和调节中起作用，因此定义了分子的一部分， 包含活性位点和调节位点。我们还将研究 通过研究同位素分配和定位酶的机制 同位素交换，这将提供关于以下步骤的速率限制步骤的信息： 机制我们还将进行pH值研究，以确定这些组 必须质子化或非质子化才能催化或调节。通过 做这些研究与那些化学衍生研究相结合 上面将区分重要的残基。使用该信息 再加上其他PFK的知识，我们将诱变某些残留物，表达 分离突变蛋白，研究突变氨基酸对蛋白质合成的影响， 酸的催化或调节作用。通过这种方式，我们应该能够 预测催化机制应该是什么。我们亦会研究 PFK的结构与功能有关。这些条件也 确定四聚体解离成二聚体的条件，或 单体。PFK的圆二色性光谱将在以下条件下测定： 它处于其天然状态，而不是磷酸化状态。我们将 还注意到当运行d-PFK和pd-PFK时光谱的差异。 最后，将利用重组PFK进行结晶研究。 开始于我们已经开发的各种形式的PFK（n-PFK， d-PFK、pn-PFK、pd PFK、o-PFK）的能力进行测试， 在底物、产物和效应物存在下结晶。

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.