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.
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