Biochemical characterization of GTP binding properties of Human phosphoglucose isomerase

• Main Authors: Jia-Yun Lin, 林嘉筠
• Other Authors: Meng-Hsiao Meng
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/43669985603978907524

碩士 === 國立中興大學 === 生物科技學研究所 === 96 === Phosphoglucose isomerase (PGI) is a multifunctional protein. Within cell, it catalyzes the reversible isomerization between glucose-6-phosphate and fructose-6-phosphate in glycolysis and gluconeogenesis pathways. Outside the cells, it moonlights as autocrine mobility factor (AMF) involved in cancer metastasis, neurolukin supporting the survival of specific neurons, and maturation factor mediating the differentiation of myeloid leukemic HL-60 cells to terminal monocytic cells. In this study, we accidentally found that human PGI (hPGI) could bind to [α-32P]GTP and form a covalent complex after UV irradiation. To assure the binding was resulted from hPGI but not possible contaminants in the protein sample, the hPGI was purified by using a GTP-sepharose column. Experiments showed that hPGI does bind to GTP-sepharose and can be eluted with 5 mM GTP from the column, proving the GTP-binding ability of hPGI. GTP is the best competitor for [α-32P]GTP in comparing with other nucleotides, suggesting that hPGI prefers to bind GTP than other nucleotides. To investigate whether the GTP-binding ability is unique to hPGI, the GTP-binding ability of E. coli PGI was also analyized. The results showed that the GTP binding ability of E. coli PGI was much weaker than that of hPGI. Enzymatic assay showed that GTP has insignificant effect on the isomerization activity of hPGI. TLC analysis and Enzyme-linked assay showed that hPGI could not hydrolyze GTP. Furthermore, the LC/MS/MS results showed that the peptide sequences of hPGI, which bind to GTP, include 58