| Summary: | 碩士 === 國立中興大學 === 食品暨應用生物科技學系所 === 103 === Uridine phosphorylase(upase)is an important enzyme in the nucleotide salvage pathway. The substrates of this enzyme include uridine,phosphate,uracil and ribose 1-phosphate. Previous studies showed that E.coli upase seems apparently destabilized in the presence of adenosine triphosphate(ATP). The possible mechanism of this phenomenon may come from the interaction between ATP and the intermediate state of upase. In addition,this interaction may change the folding pathway of upase which can possibly be correlated to the the biological function of this enzyme. To understand the detail mechanism of this interaction,we have designed a series of experiments to study the effect of ATP on the intermediates of upase.
First,we tried to purify upase in order to rule out factors that may complicate the interaction between ATP and upase. Cloning experiments successfully incorporated the gene of upase into pET28b plasmid. We then over-expressed upase in E.coli BL21(DE3). By combining DEAE sepharose Fast Flow and His-tagged affinity chromatography,we purified the E.coli upase as dominating protein. Finally,We measured the thermodynamic stability of upase in the presence or absence of ATP by pulse proteolysis. The results show that ATP apparently destabilizes the E.coli upase .
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