Characterization of 5’-nucleotidase by using ultraviolet-visible spectrometry

• Main Authors: Chi,Yi-Lun, 紀懿倫
• Other Authors: Tzeng,Huey-Fen
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/23179516722724570336

碩士 === 國立暨南國際大學 === 應用化學系 === 96 === The 5’-nucleotidases (5’-NT) are involved in regulation of various physiologically active nucleosides, nucleotides, and their analogues in organs. Therefore 5’-NT maintains balanced nucleotide pools, so it is one of the important phosphohydrolases. The 5’-NT catalyzes the dephosphorylation of AMP to form adenosine and phosphate ion, and IMP to form inosine and phosphate. Herein, phosphate ion was derivatized, and then determined by using ultraviolet-visible (UV-VIS) spectrometry to characterize the 5’-NT from insect cell Sf9, normal liver cell Clone9 and human hepatocellular carcinoma Hep G2. The kinetics of Sf9 5’-NT-catalyzed AMP dephosphorylation followed Hill equation suggested that 5’-NT was regulated by allosteric effectors. The maximum catalytic velocity and Km of Sf9 5’-NT were 68.4 nmol/min/mg and 46.9 mM, respectively. The 5’-NT from Clone9 and Hep G2 followed Michaelis Menten kinetics. Hep G2 5’-NT has vmax,AMP 11.7 nmol/min/mg with Km,AMP 57 M and vmax,IMP 11.7 nmol/min/mg with Km,IMP, 470 M. Clone9 5’-NT has vmax,IMP 10.5 nmol/min/mg and Km,IMP 330 M implied that most 5’-NT activity catalyzing IMP dephosphorylation was from cytosolic 5’-NT. Compared with previous data the location of major 5’-NT activity could be determined by the optimal pH of the enzyme catalyzed reaction. The pH optimum of Hep G2 at pH 8.0 with vmax,AMP 12.8 nmol/min/mg suggested that the main 5’-NT activity for AMP dephosphorylation was contributed from ecto 5’-NT, and that at pH 6.5 with vmax,IMP 12.1 nmol/min/mg suggested that the activity for IMP dephosphorylation was from cytosolic 5’-NT.