Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36189
標題: 人類6-磷酸葡萄糖異構酶與鳥糞嘌呤核苷三磷酸鹽結合之生化特性探討
Biochemical characterization of GTP binding properties of Human phosphoglucose isomerase
作者: 林嘉筠
Lin, Jia-Yun
關鍵字: phosphoglucose isomerase
6-磷酸葡萄糖異構酶鳥糞嘌呤核苷三磷酸鹽
GTP
出版社: 生物科技學研究所
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(1933) Uber phosphorylierung und Dephosphorylierung. Bildung der naturlichen Hexosemonophosphorsaure aus ihren Komponenten. Biochem.Z. 262:137. Matsumoto, I., Staub, A., Benoist, C. & Mathis, D. (1999) Arthritis provoked by linked T and B cell recognition of a glycolytic enzyme. Science 286:1732–1735. McMorris FA, Chen TR, Ricciuti F, Tiscfield J, Creagan R, Ruddle FH. (1973) Chromosome assignments in man of genes for two hexosephosphate isomerases. Science 179:1129-1131. Mizrachi, Y. (1989) Neurotrophic activity of monomeric glucophospho- isomerase was blocked by human immunodeficiency virus (HIV-1) and peptides from HIV-1 envelope glycoprotein. J Neurosci Res 23:217. Monica, S., Dennis, R., Burton, Henrik, J., Ditzel. (2001) Autoantibodies to GPI in rheumatoid arthritis: Linkage between an animal model and human disease. Nature immunolol 2:746. Nabi IR., Watanabe H., and Raz A. (1990) Identification of B16-F1 melanoma autocrine motility-like factor receptor. Cancer Res 50:409. Patel, P. S., Raval, G. N., Rawal, R. M., Patel, G. H., Balar, D. B., Shah, P. M. & Patel, D. D. (1995) Comparison between serum levels of carcinoembryonic antigen, sialic acid and phosphohexose isomerase in lung cancer. Neoplasma 42:271–274. Salas, M., Vinuela, E. and Sola, A. (1965) Spontaneous and enzymatically catalyzed anomerization of glucose-6-phosphate and anomeric specificity of related enzymes. J. Biol. Chem. 240:561. Schweins, T., Geyer, M., Scheffzek, K., Warshel, A., Kalbitzer, H., & Wittinghofer, A. (1995) Substrate-assisted catalysis as a mechanism for GTP hydrolysis of p21ras and other GTP-binding proteins. Nat. Struct. Biol. 2:36–44. Shimizu, K., Tani, M., Watanabe, H., Nagamachi, Y., Niinaka, Y., Shiroishi, T., Ohwada, S. & Raz, A. (1999) The autocrine motility factor receptor gene encodes a novel type of seven transmembrane protein. FEBS Letters 456:295–300. Tanaka, N., Haga, A., Uemura, H., Akiyama, H., Funasaka, T., Nagase, H., Raz, A., & Nakamura, K. T. (2002) Inhibition Mechanism of Cytokine Activity of Human Autocrine Motility Factor Examined by Crystal Structure Analyses and Site-directed Mutagenesis Studies. J. Mol. Biol. 318:985–997. Tor, J., Segura, R. M., Pascual, C., Vilaseca, J., Guarner, M. L., Schwartz, S. (1981) Determination of serum activity of glucose phosphate isomerase and glutathione reductase in intra and extra hepatic cholestasis. Med Clin (Barc) 77:236. Tor, J., Pascual, C., Segura, R. M., Vilaseca, J. and Schwartz, S. (1982) Value of glutathione reductase and glucosephosphate isomerase measurements in infectious diseases. Rev Clin Esp 164:15. Tsutsumi, S., Gupta, S. K., Hogan, V., Collard, J. G. & Raz, A. (2002) Activation of small GTPase Rho is required for autocrine motility factor signaling. Cancer Research 62:4484–4490. Walker JI, Faik P, Morgan MJ. (1990) Characterization of 5’ end of the gene for human glucose phosphate isomerase. Genomics 7:638-643. Walker JI, Morgan MJ, Faik P. 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摘要: 6-磷酸葡萄糖異構酶 ( Phosphoglucose isomerase,PGI ) 是一個多功能的酵素。廣泛存在於各種細胞內,主要參與醣解作用及醣質新生作用,其功能在催化6-磷酸葡萄糖與6-磷酸果糖間可逆性的轉換。在細胞外,PGI是一種腫瘤自泌移動因子和腫瘤轉移有關,也是一種神經白介素和促分化成熟介子,分別具有支持脊髓及感覺神經元存活的能力和誘導白血病細胞HL-60成為單核白血球。本實驗發現人類PGI與[α-32P]GTP在UV短暫照射後,會形成共價複合體,顯示人類PGI具有結合GTP的能力。為了確保UV照射後自動顯影上訊號的確就是PGI與[α-32P]GTP的結合,而非分子量相近的微量雜蛋白質與[α-32P]GTP的結合,我們將PGI通過GTP-sepharose管柱,之後再以GTP沖提,結果PGI確實能結合到管柱上,再被沖洗下來,此證實PGI確實能結合GTP。利用其他種核苷酸競爭試驗發現,核苷酸中以GTP最能與[α-32P]GTP競爭,暗示PGI對GTP有最強的結合力。為了知道PGI與GTP結合是否為人類PGI所獨有的特性,我們利用大腸桿菌PGI測試其與GTP結合的能力,發現大腸桿菌PGI與GTP結合能力並不如人類PGI。在利用酮糖化學呈色法,發現GTP對PGI轉換6-磷酸葡萄糖的能力並沒有顯著影響。利用薄層層析法 (TLC) 與酵素連結法分析PGI與GTP結合後,觀察GTP水解反應的發生,結果發現PGI不具水解GTP的能力。我們進一步利用串聯質譜儀作胺基酸序列分析,得知在PGI上參與與GTP結合的肽胜片段包括N端第58個到第66個的NLVTEDVMR、第29個到第36個的LFDANKDR以及第67個到第75個的MLVDLAKSR。
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
URI: http://hdl.handle.net/11455/36189
其他識別: U0005-2506200811280000
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