Purification and Characterization of Bovine Liver Uridine Phosphorylase

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Avant, Jamie Danielle
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Middle Tennessee State University
Uridine phosphorylase (UPP) is an enzyme that catalyzes the reversible phosphorylytic cleavage of uridine to uracil and αribose–1–phosphate.1 A strong interest has been expressed in the enzyme in recent years because of the recognition of its many medicinal applications. Naturally occurring uridine has been shown to exhibit a cytoprotective effect against the toxicity associated with chemotherapy treatments for both oncological and infectious diseases.2 Inhibition of UPP drastically slows the breakdown of uridine and therefore raises plasma uridine levels in mammals. Determining a purification scheme is the first step to acquiring a pure enzyme that can be studied in the pursuit of the development of stronger and more specific UPP inhibitors. The purification scheme outlined in this thesis consisted of the following steps: ammonium sulfate precipitation, Mono Q ion exchange chromatography, Sephacryl S100 size exclusion chromatography, and hydroxyapatite chromatography. UPP was purified 64–fold with 4.01% final yield. The initial extract showed a specific activity of 0.41 nmoles/min/mg, which increased to 26.51 nmoles/min/mg after the final purification step. Additionally, the purified enzyme was found to exhibit cleaving activity for uridine, thymidine and inosine in the ratio of 5:1:0.
Beef, Bovine, Liver, Nucleoside, Phosphorylase, Uridine