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研究生:曾靖涵
研究生(外文):Ching-Han Tseng
論文名稱:綠竹筍蔗糖磷酯合成酶之生化學研究
論文名稱(外文):Biochemical Studies of Sucrose Phosphate Synthase from bamboo shoot
指導教授:李平篤李平篤引用關係
指導教授(外文):Ping-Du Lee
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:綠竹筍蔗糖磷酯合成酶異位調控
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蔗糖磷酯合成酶(sucrose phosphate synthase ) 是植物蔗糖生合成的一個重要調控酵素,本論文以綠竹筍為材料,粗抽液經過陰離子交換層析 (DEAE-Sephacel chromatography)、24% PEG 沉澱、親和層析 (w-aminohexyl-Sepharose 4B chromatography)、快速蛋白質分離層析 (FPLC/Mono Q HR 5/5 chromatography) 純化步驟處理後,所得到之蔗糖磷酯合成酶,其純化倍率為 46.7,而回收率為0.12%。
純化出之綠竹筍蔗糖磷酯合成酶的酵素原態及單元體分子量約為 540 及 130-140 kD,因此竹筍 SPS 應為同質四元體形結構。又 SPS 之最適反應 pH 為7.5,而最適反應溫度為 37℃。其酵素活性會受 AMP、IMP、G1P的活化,但會受到鋅、鈷、汞、亞錳離子及 UTP、GTP、Glucose、Cibacron blue 3GA、1-Deoxynojirimycin 的抑制。綠竹筍蔗糖磷酯合成酶並不會受 G6P 及 Pi 的異位調控,對基質 Fru-6-P 之 Km 值為 2.74 mM,而對基質 UDPG 之Km 值則為 38.4 mM。
Sucrose phosphate synthase (SPS) is an important regulatory enzyme of sucrose biogenic reaction in the plant. In this study, SPS was purified from bamboo shoots by the steps of DEAE-Sephacel chromatography, 24% PEG precipitation, w-aminohexyl-Sepharose 4B chromatography, and Mono Q HR 5/5 chromatography. The purification fold was 46.7 and the yield was 0.12%.
The native and subunit molecular mass of SPS were estimated to be 540 and 130-140 kD respectively and may be a homotetramer in nature. The optimal pH and temperature of SPS-catalyzed reation were 7.5 and 37℃, respectively. Bamboo shoot SPS was slightly activated by AMP, IMP, G1P and inhibited by Zn2+, Co2+, Hg2+, Mn2+, UTP, GTP, Glucose, Cibacron blue 3GA, and 1-deoxynojirimycin. SPS from bamboo shoots was not allosterically regulated by glucose 6-phosphate and inorganic phosphate. The Km values for fructose 6-phosphate and UDPG of SPS were 2.74 mM and 38.4 mM, respectively.
第一章 緒論
1.1 蔗糖的生合成與分解……………………………………………… 1
1.2 蔗糖磷酯合成酶之生化特性及生理意義…………………………… 3
1.3 蔗糖磷酯合成酶活性之調控………………………………………….4
1.4 蔗糖磷酯合成酶激酶(SPS kinase) 與蔗糖磷酯合成酶之蛋白質磷酯 (SPS-protein phosphatase) …………………………………………5
1.4.1 蔗糖磷酯合成酶激酶(SPS kinase)……………………………… 5
1.4.2 蔗糖磷酯合成酶之蛋白質磷酯脢 (SPS-protein phosphatase) 6
1.5 蔗糖磷酯合成酶的分類……………………………………………… 7
1.6 蔗糖磷酯合成酶的分子生物學研究………………………………… 8
1.7 蔗糖磷酯合成酶的酵素複合體……………………………………….9
1.8 蔗糖磷酯合成酶之其他研究…………………………………………10
1.9 實驗緣起………………………………………………………………11
第二章 材料與方法……………………………………………………… 12
2.1 材料、藥品與儀器……………………………………………………12
2.2 蛋白質定量法…………………………………………………………13
2.3 蔗糖磷酯合成酶活性測定……………………………………………15
2.4 蔗糖合成脢之酵素活性分析…………………………………………18
2.5 電泳檢定系統………………………………………………… …… 19
2.5.1 原態膠體電泳………………………………………………………19
2.5.2 SDS-膠體電泳………………………………………………… … 23
2.6 Coomassie Brilliant Blue R-250 (CBR) 蛋白質染色法………26
2.6.1 膠片乾燥法…………………………………………………………27
2.7 蔗糖磷酯合成酶之純化………………………………………………28
2.7.1 酵素的粗抽…………………………………………………………28
2.7.2 DEAE-Sephacel 層析………………………………………………29
2.7.3 w-aminohexyl-Sepharose 4B 管柱層析…………………………31
2.7.4 快速蛋白質液相層析………………………………………………33
2.7.4.1 Mono Q HR 5/5 chromatography………………………………34
2.7.4.2 Superose 6 HR 10/30 chromatography………………………36
2.8 酵素生化性質分析……………………………………………………38
2.8.1 最適反應溫度………………………………………………………38
2.8.2 最適 pH 值測定……………………………………………………39
2.8.3 葡萄糖 6-磷酯 (G-6-P) 對酵素活性的影響……………………40
2.8.4 無機磷酸鹽 (Pi) 對酵素活性的影響……………………………40
2.8.5 金屬離子對酵素活性的影響………………………………………41
2.8.6 代謝物質對酵素活性的影響………………………………………42
2.8.7 蔗糖磷酯合成酶之動力學研究……………………………………43
2.8.8 Cibacron blue 3GA 及 1-Deoxynojirimycin 對酵素活性的影響44
第三章 結果與圖表……………………………………………………… 45
3.1 酵素純化方法的探討…………………………………………………45
3.2 蔗糖磷酯合成酶純化結果……………………………………………45
3.2.1 純化步驟之流程……………………………………………………46
3.2.2 DEAE-Sephacel chromatography (batch)……….…………… 47
3.2.3 w-aminohexyl-Sepharose 4B chromatography…………………49
3.2.4 Mono Q HR 5/5 chromatography…………………………………53
3.2.5 各個純化步驟的電泳分析結果……………………………………57
3.2.6 蔗糖磷酯合成酶的純化效率………………………………………60
3.3 蔗糖磷酯合成酶原態分子量的測定…………………………………62
3.4 綠竹筍蔗糖磷酯合成酶的生化性質分析……………………………65
3.4.1 最適反應溫度………………………………………………………65
3.4.2 最適反應 pH……………………………………………………… 65
3.4.3 不同金屬離子的影響………………………………………………65
3.4.4 不同代謝物質的影響………………………………………………66
3.4.5 G6P、Pi的影響…………………………………………………… 66
3.4.6 酵素動力學研究……………………………………………………66
3.4.7 Cibacron blue 3GA 及 1-deoxynojirimycin 的影響…………67
第四章 討論與展望……………………………………………………… 78
參考文獻……………………………………………………………………82
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