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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 莊榮輝 | |
| dc.contributor.author | Hsin-Chieh Wang | en |
| dc.contributor.author | 王信傑 | zh_TW |
| dc.date.accessioned | 2021-06-15T02:21:59Z | - |
| dc.date.available | 2009-08-20 | |
| dc.date.copyright | 2009-08-20 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-08-19 | |
| dc.identifier.citation | 莊榮輝 (1988) 水稻蔗糖合成脢之研究。博士論文。國立台灣大學農業化學所，台北。
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43460 | - |
| dc.description.abstract | 植物螯合素合成酶 (PCS, EC 2.3.2.15) 利用穀胱甘肽 (GSH) 當作基質進行 植物螯合素 (PCs) 合成。根據先前已發表論文得知 PCS 在生物體內是處於持續表現狀態，因此推測其調控機制可能經由後轉譯修飾。的確，將阿拉伯芥 PCS 序列 (AtPCS1) 經軟體分析後得知其上包含數個可能受到磷酸化修飾位置。而 In vitro 實驗也證明 PCS 活性會受到 casein kinase 2 (CK2) 磷酸化與 calf intestine alkaline phosphatase (CIAP) 去磷酸化調控。當 PCS 被 CIAP 去磷酸化後活性會下降，再經 CK2 磷酸化後則活性恢復。經由放射性點突變實驗證明 AtPCS1 磷酸化位置就在 Thr49。當 Thr49 突變成 Ala 後，AtPCS1 即無法被 CK2 磷酸化，而且活性也明顯下降。另外利用軟體所預測 AtPCS1 3D 立體結構顯示 PCS的 Arg183 在空間位置上非常接近 Thr49，將 Arg183 突變成 Ala 後發現 AtPCS1 仍然可以被 CK2 所磷酸化，但是活性卻明顯下降，推測磷酸化 Thr49 與 Arg183 兩者間可能會相互作用來形成 second substrate-binding site。另一方面，本文亦發現 AtPCS1 磷酸化必須要有 Cd 存在，而且此磷酸化現象會受到基質 GSH 抑制。若是單以 AtPCS1 N 端催化活性區重組蛋白 (AtPCS1-N) 進行相關實驗，發現其活性受到 Cd 與磷酸化的影響更加明顯，但是 AtPCS1-N 磷酸化卻不受 Cd 或 GSH 影響，因此推論 AtPCS1 C-domain 對於 Cd 結合與磷酸化調控佔有重要地位。除此之外，經由酵素動力學實驗結果顯示對於基質 GSH 而言，AtPCS1呈現出 S 形曲線 (sigmoidal curve)，而AtPCS1-N 呈現的則是典型 Michaelis-Menten 動力學圖形 (hyperbolic curve)。再經原態分子量測定發現 AtPCS1 為二元體，AtPCS1-N 為單元體，這些結論都強烈指出 C-domain 可能參與 AtPCS1 四級結構的形成，並且在蛋白質磷酸化反應與活性調控部份扮演重要角色。 | zh_TW |
| dc.description.abstract | Phytochelatin synthase (PCS, EC 2.3.2.15) uses glutathione (GSH) as its substrate to catalyze the synthesis of heavy metal-binding peptides, known as phytochelatins (PCs). PCS has been described as a constitutive enzyme that may be controlled by post-translational modifications. Indeed, we found that the amino acid sequence of PCS from Arabidopsis (AtPCS1) contains several protein phosphorylation motifs. In vitro experiments demonstrate that PCS activity is increased following phosphorylation by casein kinase 2 (CK2), and it decreases following treatment with alkaline phosphatase. Site-directed mutagenesis at several amino acids on AtPCS1 indicates that Thr 49 is the site for phosphorylation; consistent with this idea, the mutant AtPCS1(T49A) cannot be phosphorylated, and its activity is significantly lower than that of the wild-type enzyme. In the predicted three-dimensional structure of AtPCS1, Arg 183 is close to Thr 49. The mutant AtPCS1(R183A) can be phosphorylated, but it shows much lower catalytic activity than the wild-type protein. Interestingly, a second substrate-binding site forms as a result of the interaction of these two amino acids. We propose that Arg 183 interacts with the phosphorylated Thr 49 residue to give the active site of PCS a distinctive shape. Furthermore, the phosphorylation of AtPCS1 by CK2 is dependent on Cd and is inhibited by GSH. The N-terminal catalytic domain of AtPCS1 was expressed (AtPCS1-N), and its catalytic activity was found to be even more sensitive to Cd or phosphorylation status than to that of the full-length enzyme. However, unlike AtPCS1, AtPCS1-N phosphorylation is not controlled by Cd or GSH, indicating a role for the C-domain in Cd-binding and regulation of phosphorylation. In addition, kinetic studies showed that the full-length AtPCS1 activity has a sigmoidal dependence on GSH, and it is estimated to be a dimer, whereas AtPCS1-N was present only in monomeric form. The C-terminal domain may thus participate in the formation of the quaternary structure of AtPCS1, and it may play a regulatory role in protein phosphorylation, forming a competent active site that can accommodate its substrates. | en |
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| dc.description.tableofcontents | 中文摘要 1
Abstract 2 第一章 緒 論 3 1.1 環境中的重金屬污染嚴重 3 1.1.1 台灣有嚴重的重金屬污染問題 3 1.1.2 重金屬的一般定義 3 1.1.3 重金屬對生物體的傷害極大 4 1.1.4 鎘對人體所造成的傷害 4 1.1.5 如何清除環境重金屬污染 5 1.1.6 以生物復育法清除重金屬污染 5 1.2 生物抗重金屬機制 7 1.2.1 鎘與鋅的化性相似 7 1.2.2 金屬硫蛋白 (metallothionein) 7 1.2.3 植物螯合素 (phytochelatin) 8 1.2.4 細胞對重金屬的其它解毒機制 10 1.3 植物螯合素生合成 11 1.3.1 植物螯合素與鎘會形成複合物 13 1.3.2 鎘複合物是經由液泡膜上的HMT1蛋白質運送至液泡內 13 1.4 植物螯合素合成酶 15 1.4.1 植物螯合素合成酶之純化 15 1.4.2 植物螯合素合成酶之基因序列 15 1.4.3 植物螯合素合成酶之作用機制 16 1.4.4 植物螯合素合成酶是一種dipeptidyltransferase 21 1.4.5 植物螯合素合成酶屬於papain superfamily protease 25 1.5 實驗緣起 27 第二章 材料與方法 30 2.1 酵母菌培養 30 2.1.1菌種 30 2.1.2 洋菜膠培養法 30 2.1.3 液態培養法 31 2.1.4 濾紙鎘擴散培養法 32 2.2 酵母菌的粗抽取液製備 34 2.2.1 玻璃砂萃取法 34 2.3 阿拉伯芥培養 35 2.3.1 植物材料 35 2.3.2 培養基配製 35 2.3.3 種子之表面消毒與低溫處理 36 2.3.4 種子之無菌培養 37 2.3.5 種子之土壤培養 37 2.3.6 種子之採集 37 2.3.7 植株之採集 37 2.4 阿拉伯芥的粗抽取液製備與硫酸銨沈澱 37 2.4.1 果汁機均質法 37 2.5 一般化學分析法 40 2.5.1 蛋白質定量法 40 2.5.2 胺基酸組成分析法 41 2.5.3 膠體內 trypsin 水解法 42 2.5.4 質譜儀分析 44 2.6 生物化學檢定法 44 2.6.1 鎘離子的測定 44 2.6.2 硫醇基的測定 45 2.6.3 酸不穩定硫離子的測定 46 2.7 管柱層析法 48 2.7.1 膠體過濾法 48 2.7.2 Hydroxylapatite (HA) 層析法 50 2.7.3 離子交換法 51 2.7.4 金屬螯合層析法 52 2.7.5親和性層析法 (Protein A-Sepharose CL-4B) 53 2.7.6 高效液相層析法 (HPLC) 54 2.8 電泳檢定法 56 2.8.1 原態膠體電泳 (native-PAGE) 56 2.8.2 SDS膠體電泳 59 2.8.3 Tricine SDS-PAGE 60 2.8.4二維雙向電泳 (2-dimensional electrophoresis, 2-DE) 64 2.8.4.1 等電點焦集法 (isoelectric focusing, IEF) 64 2.8.4.2 二維電泳：SDS-PAGE 66 2.8.5 膠體染色法 67 2.8.5.1 Coomassie Brilliant Blue R (CBR) 染色法 67 2.8.5.2硝酸銀染色法 68 2.8.5.3 Ponceau S 染色法 69 2.8.6 膠片乾燥法 70 2.8.7 蛋白質電泳轉印法 70 2.9 植物螯合素合成酶純化 72 2.9.1 PCS 活性分析 72 2.9.1.1 HPLC分析法 72 2.9.2 Casein Kinase II (CK2) 活性分析法 73 2.10 單株抗體之製備 75 2.10.1 小白鼠免疫 75 2.10.2 細胞融合 76 2.10.3 細胞保存法 80 2.10.3.1細胞冷凍法 80 2.10.3.2 細胞解凍法 81 2.10.4 單株抗體的生產 82 2.10.5免疫球蛋白之純化 82 2.11 酵素免疫分析法 84 2.12 酵素免疫染色法 86 2.13 電子顯微鏡法 89 2.14 阿拉伯芥植物螯合素合成酶重組蛋白製備 91 2.14.1 載體 DNA 91 2.14.2 大腸桿菌菌株 91 2.14.3 專一性引子設計 (specific primers) 92 2.14.4 聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 92 2.14.5 洋菜膠體電泳 (agarose gel electrophoresis) 94 2.14.6 DNA 片段的分離與純化 95 2.14.7 DNA 的定量 96 2.14.8 T-A cloning 96 2.14.8.1 A-tailing 96 2.14.9 DNA 接合反應 (ligation) 97 2.14.10 質體之轉形 (transformation) 97 2.14.11 藍白篩選 98 2.14.12 質體快速檢定 98 2.14.13 質體 DNA 之小量製備法 99 2.14.14 DNA 限制酶分析法 100 2.14.15 DNA 序列分析 100 2.14.16 表現載體之建構 100 2.14.16.1 表現載體之選擇 101 2.14.16.2 表現載體宿主菌種保存 101 2.14.17 重組蛋白之表現與檢定 101 2.14.17.1 重組蛋白大量表現之誘導 101 2.14.18 重組蛋白之純化 102 第三章 結果與討論 104 3.1酵母菌可產生金屬光澤現象 104 3.1.1鎘會誘導酵母菌產生金屬光澤物質 104 3.1.2酵母菌產生金屬光澤的可能原因 105 3.2利用 SEM 觀察加鎘培養的酵母菌細胞 106 3.2.1加鎘處理後之 area 1、area 2 細胞表面出現微小顆粒 106 3.2.2 area 3 細胞與正常細胞外觀相似 106 3.2.3細胞外鎘結合物可被緩衝液輕易洗下 107 3.3 鎘結合物 H6 的分離純化 107 3.3.1比較各區之鎘含量與酸不穩定硫離子含量 107 3.3.2利用膠體過濾法純化 H6 108 3.3.3利用逆相高效液相層析法純化 H6 109 3.3.4利用快速液相層析法與薄層層析法純化 H6 110 3.4 植物螯合素合成酶的純化 111 3.4.1植物螯合素合成酶的活性分析 111 3.4.2生物體內植物螯合素合成酶的純化 113 3.4.2.1使用膠體過濾層析法純化 (Sephacryl S-300) 113 3.4.2.2 使用 Hydroxylapatite 層析法純化 113 3.4.2.3使用陰離子交換層析法純化 (DEAE Sephacel) 114 3.5 阿拉伯芥 AtPCS1 重組蛋白製備 114 3.5.1表現載體之建構 115 3.5.2重組蛋白之表現及檢定 115 3.6 植物螯合素合成酶單株抗體製備 116 3.6.1抗原製備方法 117 3.6.2抗體篩選方法 117 3.6.3 利用 proteinase K 切解 AtPCS1 片段進行抗體製備 117 3.7 植物螯合素合成酶催化機制探討 119 3.7.1 IAA 會抑制植物螯合素合成酶的作用 119 3.7.2 利用 limited proteolysis 研究植物螯合素合成酶的催化機制 120 3.7.3 阿拉伯芥 AtPCS1 含有蛋白質激酶專一性辨識的保守性序列 121 3.7.4 重組蛋白 AtPCS1 與 AtPCS1-N 在 E. coli 內磷酸化現象 122 3.7.5 重組蛋白 AtPCS1 與 AtPCS1-N 可以被 CK2 磷酸化 124 3.7.6 重組蛋白 AtPCS1 與 AtPCS1-N 可以被 CIAP 去磷酸化 125 3.7.7 重組蛋白 AtPCS1 磷酸化反應受 Cd 調控並且被 GSH 所抑制 126 3.7.8 AtPCS1 活性會受到磷酸化與去磷酸化反應調控 127 3.7.9 AtPCS1 與 AtPCS1-N 之酵素動力學研究 128 3.8 植物螯合素合成酶磷酸化位置與催化機制模型探討 130 3.8.1 利用定點突變技術確認 AtPCS1 上磷酸化位置在 Thr49 130 3.8.2 AtPCS1 催化機制模型預測 (一) 131 3.8.3 AtPCS1 催化機制模型預測 (二) 133 3.8.4 利用 AtPCS1 單株抗體進行相關催化機制研究 135 3.9 阿拉伯芥內生性 At-PCS 純化與生化性質分析 137 第四章 未來研究方向 138 4.1 本論文主要成果 138 4.2 未來研究方向 139 圖表集 142 參考文獻 208 | |
| dc.language.iso | zh-TW | |
| dc.subject | 蛋白質磷酸化 | zh_TW |
| dc.subject | 植物螯合素 | zh_TW |
| dc.subject | 植物螯合素合成酶 | zh_TW |
| dc.subject | protein phosphorylation | en |
| dc.subject | phytochelatin synthase | en |
| dc.subject | Phytochelatin | en |
| dc.title | 植物螯合素合成酶催化機制研究 | zh_TW |
| dc.title | Studies on the Catalytic Mechanism of Phytochelatin Synthase | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 林棋財,陳翰民,吳建興,楊健志,張世宗 | |
| dc.subject.keyword | 植物螯合素,植物螯合素合成酶,蛋白質磷酸化, | zh_TW |
| dc.subject.keyword | Phytochelatin,phytochelatin synthase,protein phosphorylation, | en |
| dc.relation.page | 213 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-08-19 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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