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研究生:林嘉筠
研究生(外文):Jia-Yun Lin
論文名稱: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
論文名稱(外文):Biochemical characterization of GTP binding properties of Human phosphoglucose isomerase
指導教授:孟孟孝
指導教授(外文):Meng-Hsiao Meng
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:57
中文關鍵詞:6-磷酸葡萄糖異構酶鳥糞嘌呤核苷三磷酸鹽
外文關鍵詞:phosphoglucose isomeraseGTP
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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
目錄
中文摘要 Ⅰ
英文摘要 Ⅱ
目錄 Ⅳ
表目錄 Ⅵ
圖目錄 Ⅶ
第一章、 前言 1
第一節、 6-磷酸葡萄糖異構酵素之特性 1
第二節、 不同物種間6-磷酸葡萄糖異構酵素之相似性 4
第三節、 6-磷酸葡萄糖異構酵素之結構與催化位 4
第四節、 研究動機與目的 5
第二章、 材料與方法 6
第一節、 材料介紹 6
第二節、 人類6-磷酸葡萄糖異構酵素的大量製備及純化 6
第三節、 大腸桿菌6-磷酸葡萄糖異構酵素的大量製備及純化 9
第四節、 蛋白質定量 11
第五節、 蛋白質電泳分析 11
第六節、 6-磷酸葡萄糖異構酵素活性分析 12
第七節、 GTP水解活性分析 14
第八節、 6-磷酸葡萄糖異構酵素與GTP結合力分析 16
第九節、 甲醛交叉結合 18
第三章、 結果 20
第一節、 重組6-磷酸葡萄糖異構酵素的表達與純化 20
第二節、 6-磷酸葡萄糖異構酵素與GTP結合力分析 21
第三節、 不同核苷酸與人類6-磷酸葡萄糖異構酵素結合能力的探討 22
第四節、 人類6-磷酸葡萄糖異構酵素活性分析 22
第五節、 GTP水解活性分析 23
第六節、 人類6-磷酸葡萄糖異構酵素結合GTP之結合定位探討 24
第四章、討論 26
第五章、參考文獻 30
表 37
圖 40


表目錄
表一、 不同物種間的6-磷酸葡萄糖異構酵素序列相似性 37
表二、 質譜分析結果 38
表三、 G蛋白重要motif 39


















圖目錄
圖一、 6-磷酸葡萄糖異構酵素所催化之異構反應 40
圖二、 6-磷酸葡萄糖異構酵素演化親緣關係圖 41
圖三、 人類6-磷酸葡萄糖異構酵素的X光結晶圖 42
圖四、 人類6-磷酸葡萄糖異構酵素蛋白質純化步驟之10 % SDS-PAGE蛋白質電泳分析圖 43
圖五、 大腸桿菌6-磷酸葡萄糖異構酵素蛋白質純化步驟之10 % SDS-PAGE蛋白質電泳分析圖 44
圖六、 利用UV-cross linking偵測人類6-磷酸葡萄糖異構酵素與GTP結合之能力 45
圖七、 偵測人類與大腸桿菌6-磷酸葡萄糖異構酵素與GTP結合之差異 46
圖八、 利用GTP sepharose偵測人類6-磷酸葡萄糖異構酵素與GTP結合能力 47
圖九、 利用GTP-sepharose偵測人類6-磷酸葡萄糖異構酵素與GTP結合能力之10 % SDS-PAGE蛋白質電泳分析圖 48
圖十、 參照組 (Blank) 的ITC分析圖 49
圖十一、 實驗組 (Sample) 的ITC分析圖 50
圖十二、 利用不同種的核甘酸與[α-32P]GTP競爭6-磷酸葡萄糖異構酵素試驗 51
圖十三、 利用TLC分析人類6-磷酸葡萄糖異構酵素隨時間變化 (Time course) 水解GTP能力之測試 52

圖十四、 利用TLC分析人類6-磷酸葡萄糖異構酵素水解GTP能力與酵素量之關係 53
圖十五、 離子強度對GTP水解活性之影響 54
圖十六、 利用Glucose、G6P與[α-32P]GTP競爭6-磷酸葡萄糖異構酵素試驗 55
圖十七、 利用E4P、G6P與[α-32P]GTP競爭6-磷酸葡萄糖異構酵素試驗 56
圖十八、 GTP與人類6-磷酸葡萄糖異構酵素結合位置之3D結構圖示 57
壹、中文參考文獻
謝嘉榮。1999。人類及大腸桿菌磷酸葡萄糖異構酵素之大量表達系統及純化方法的建立。國立中興大學生物科技學研究所碩士論文。

陳淑婷。2002。人類6-磷酸葡萄糖異構酵素之遺傳突變點對活性及穩定性的影響。國立中興大學生物科技學研究所碩士論文。

貳、西文參考文獻
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