The specific aims are purification and characterization of Uridine Phosphorylase, Thymidine Phosphorylase and dihydrouracil dehydrogenase, enzymes having a great impact on the metabolism of pyrimidines and their analogues. Purification of two pyrimidine phosphorylase is well on the way with conventional methods. We will characterize the enzymes in terms of molecular weights, isoelectric points, degree of aggregation etc. Complete kinetic analysis will be carried out iwth uridine phosphoorylase as was done with thymidine phosphorylase. Also applied will be the modern techniques such as monoclonal antibody affinity chromatography and gene cloning. Sufficient quantities and purities of enzymes will be purified for future studies, e.g., primary sequencing, X-ray crystallography. We have found that dihydrouracil dehydrogenase is widely distributed in the crude extract of various human and animal tissues. The liver enzyme is characterized by substrate inhibition, allosterism, and enzyme hysteresis, whereas extrahepatic tissues, particularly human lymphocytes, are characterized by the lack of all these properties and dihydropyrimidinase. Dihydrouracil dehydrogenase will be purified from mouse liver and characterized to confirm these findings.
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