| Summary: | 碩士 === 國立暨南國際大學 === 應用化學系 === 94 === The 5’-nucleotidaes (5’-NT) are involved in the regulation different physiological activation of nucleosides, nucleotides, and their analogues in organs. Therefore 5’-NT maintains balanced nucleotide pools, so it is one of the important phosphohydrolases. The traditional enzyme assays of 5’-NT are quite complicated, and need more sample. In order to develop non-radioactive assays to characterize the 5’-NTs from Hep G2 and Sf9, adenosine, hypoxanthine, inosine and AMP were separated by capillary zone electrophoresis. For the assay of 5’-NT, the standards were effectively separated in only 6 minutes. Addition of acetonitrile and sodium chloride to sample buffer could enhance sample stacking. The correlation coefficient of Ado、hXan and I were 0.9998, 0.9997, and 0.9993, respectively. The concentration limits of detection of Ado、hXan and I were 2.49 M、2.79M and 4.36 M, respectively. In the precision test, all relative standard deviations of migration time and area ratio were smaller than 1.60 % and 7.47 %, respectively. This method was applied to characterize the 5’-NT related to AMP metabolism. The kinetics of the 5'-NTs from Hep G2 and Sf9 followed Hill equation. It suggested that these 5’-NTs were regulated by allosteric effectors. The 5’-NT from Hep G2 showed higher affinity to AMP compared to that from Sf9; however, the latter showed a larger maximum velocity than the former.
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