REGULATION OF METABOLISM IN PARASITIC HELMINTHS

这个项目的长期目标是勾勒出监管机构 调节碳水化合物降解和能量产生的控制 猪蛔虫寄生线虫的肌肉。具体目标 1)纯化、物化和动力学表征 从猪蛔虫肌肉中提取磷酸化酶激酶；2)纯化， 磷酸果糖激酶的物理化学和动力学特性 来自蛔虫肌肉的蛋白激酶(PFK)；以及3)利用 利用蛔虫肌肉-角质层灌流系统研究其相互关系 循环AMP依赖和独立的监管系统。最基本的 从这个项目中获得的知识可以为 针对寄生虫的化疗药物的开发。这些酶将会 通过开发使用亲和和离子交换的程序进行纯化 层析、凝胶过滤和沉淀程序。的标准 纯度将通过重新层析，分析性超速离心法和 变性凝胶电泳，这将导致测定 天然分子量、亚基组成和分子量。什么时候 纯化后，磷酸化酶和PFK酶都将通过 将来自[Gamma-32P]ATP的32P掺入底物。蛔虫 磷酸化酶将作为磷酸化酶激酶的底物，并 纯化的磷酸化酶和磷酸果糖激酶将用作 PFK酶的底物。动力学表征将涉及到 蛋白质底物表观Km值和三磷酸腺苷的测定 蛋白激酶。如果PFK是环状AMP依赖的蛋白 和它同时磷酸化磷酸化的酶和 磷酸果糖激酶，这将提示糖原分解和 糖酵解通过环状AMP介导的协调控制 机制。在肌肉-角质层系统中， 环磷酸腺苷依赖系统(5-羟色胺)与环磷酸腺苷非依赖性 系统(乙酰胆碱)将在随后的不同时间段内进行 通过对磷酸化酶、磷酸化酶激活酶、 磷酸果糖激酶和PFK激酶。这些研究与 从纯化的酶上获得的那些应该提供足够的 用来推断每个控制系统在 寄生虫的整体新陈代谢。它还应该提供重要的 关于两个控制系统可能相互作用的数据。

The long term objective of this project is to delineate the regulatory controls which modulate carbohydrate degradation and energy production in the muscle of the parasitic nematode, Ascaris suum. The Specific Aims are: 1) The purification, physicochemical and kinetic characterization of phosphorylase kinase from the muscle of Ascaris suum; 2) The purification, physicochemical and kinetic characterization of the phosphofructokinase kinase (PFK kinase) from the muscle of Ascaris; and 3) The utilization of the Ascaris muscle-cuticle perfusion system to study the interrelationships of the cyclic AMP-dependent and -independent regulatory systems. The basic knowledge gained from this project could provide information for the development of chemotherapeutic agents for the parasites. The enzymes will be purified by developing procedures using affinity and ion exchange chromatography, gel filtration and precipitation procedures. Criteria of purity will be by rechromatography, analytical ultracentrifugation and denaturing gel electrophoresis, which will result in determination of native molecular weight, subunit composition and molecular weight. When purified, both phosphorylase kinase and PFK kinase will be assayed by the incorporation of 32P from [Gamma-32P]ATP into substrates. Ascaris phosphorylase will be used as a substrate for phosphorylase kinase and purified phosphorylase kinase and phosphofructokinase will be used as a substrate for PFK kinase. Kinetic characterization will involve determination of apparent Km values for protein-substrates and ATP of the protein kinases. If the PFK kinase is a cyclic AMP-dependent protein kinase and it phosphorylates both phosphorylase kinase and phosphofructokinase then this will suggest that glycogenolysis and glycolysis are coordinately controlled via a cyclic AMP-mediated mechanism. In the muscle-cuticle system, perfusion of the effectors of the cyclic AMP-dependent system (serotonin) and the cyclic AMP-independent system (acetylcholine) will be conducted for various time periods followed by analysis of activation of phosphorylase, phosphorylase kinase, phosphofructokinase and PFK kinase. Correlation of these studies with those obtained on the purified enzymes should provide sufficient information to deduce the probable role of each control system in the overall metabolism of the parasite. It should also provide significant data on the possible interaction of both control systems.