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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/39454完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 廖大修 | |
| dc.contributor.author | Wei-Jung Chen | en |
| dc.contributor.author | 陳威戎 | zh_TW |
| dc.date.accessioned | 2021-06-13T17:28:54Z | - |
| dc.date.available | 2005-10-08 | |
| dc.date.copyright | 2004-10-08 | |
| dc.date.issued | 2004 | |
| dc.date.submitted | 2004-10-04 | |
| dc.identifier.citation | 1. Moore, S. (1981) Pancreatic DNase. In The Enzymes, 3rd ed. (eds. P.D. Boyer et al.), Vol. 14, pp 281-296, Academic Press, New York.
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S., Park, J. P., and Eastman, A. (2001) The cloning, genomic structure, localization, and expression of human deoxyribonuclease II | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/39454 | - |
| dc.description.abstract | 牛胰臟去氧核糖核酸水解酶 (bpDNase) 是研究得最為透徹的一種DNase，它會水解雙股DNA，但不具序列專一性。它也是第一個被發現且以傳統蛋白質定序技術決定出胺基酸序列之DNase，目前已由前人得到其解析度達2.0 Å之X-光繞射三維晶體結構。在本論文中，我們針對bpDNase中雙硫鍵之生物功能及其N-與C-端片段參與活性蛋白摺疊之重要性進行深入的探討。
我們以定位突變法分別製備了bpDNase中非必需 (C101-C104) 及必需雙硫鍵(C173-C209) 之突變株酵素 [brDNase(C101A)、brDNase(C173A) 與brDNase(C209A)]，以研究其生化功能；另外也分析了一株具有新增第三對雙硫鍵之突變株酵素[brDNase(F192C/A217C)] 的特性。在缺乏鈣離子保護下，bpDNase與brDNase(C101A)會被胰蛋白 | zh_TW |
| dc.description.abstract | Bovine pancreatic deoxyribonuclease (bpDNase) is the best-characterized DNase which cleaves double stranded DNA with no sequence specificity. It was the first DNase discovered and sequenced with the conventional protein sequencing technique and the X-ray structure was resolved at 2.0 Å with refinement. In this dissertation, the biological functions of the disulfides in bpDNase and the involvement of its N- and C-terminal fragments in active protein folding were demonstrated.
The biochemical functions of the small non-essential (C101-C104) and the large essential (C173-C209) disulfides in bpDNase have been characterized using alanine mutants [brDNase(C101A)] and [brDNase(C173A) and brDNase(C209A)], respectively. In addition, we have characterized the effects of an additional third disulfide [brDNase(F192C/A217C)]. Without the Ca2+ protection, bpDNase and brDNase(C101A) were readily inactivated by trypsin while brDNase(F192C/A217C) remained active. With Ca2+, all forms of DNase, except for brDNase(C101A), were protected against trypsin. All forms of DNase, after being dissolved in 6 M guanidine-HCl, were fully reactivated by diluting into a Ca2+-containing buffer. However, when diluted into a Ca2+-free buffer, bpDNase and brDNase(C101A) remained inactive, but 60% of the bpDNase activity was restored with brDNase(F192C/A217C). When heated, bpDNase was inactivated at a transition temperature of 65 | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T17:28:54Z (GMT). No. of bitstreams: 1 ntu-93-D88442002-1.pdf: 1600933 bytes, checksum: b9127338f38f3547b715ab3c3bdd163b (MD5) Previous issue date: 2004 | en |
| dc.description.tableofcontents | 中文摘要……………………………… 1
英文摘要 (Abstract)……….. 3 目錄 (Table of contents)…………………………... 5 表目錄 (List of Tables)…………………………………. 8 圖目錄 (List of Figures)……………….....….. 9 縮寫表 (Abbreviations)………………………… 11 第一章、研究背景、目的與實驗構想………………. 12 第一節、去氧核糖核酸水解酶簡介………..………. 13 第二節、牛胰臟去氧核糖核酸水解酶簡介……... 16 第三節、研究目的與實驗構想………………… 20 第二章、牛胰臟去氧核糖核酸水解酶之雙硫鍵生物功能…….. 22 第一節、前言……………………………….. 23 第二節、材料與方法………………….. 24 (一) 藥品試劑…………………………... 24 (二) 儀器設備……………………... 25 (三) 定位突變法及表現質體之建構..........………. 27 (四) 重組蛋白之表現與純化……………... 31 (五) 牛胰臟去氧核糖核酸水解酶之純化……... 33 (六) 去氧核糖核酸水解酶活性分析……………... 33 (七) 蛋白質定量分析………………... 34 (八) 十二烷硫酸鈉聚丙烯醯胺膠體電泳法-銀染色法與Coomassie blue 染色….. 34 (九) 十二烷硫酸鈉聚丙烯醯胺膠體電泳法-DNase 活性染色法………. 37 (十) 硫醇基之定量……………... 39 (十一) 質體DNA切割分析…………………. 40 (十二) 圓二色偏光光譜測定……………………... 41 (十三) 胰蛋白酶對各種DNase活性之影響………………… 41 (十四) | |
| dc.language.iso | zh-TW | |
| dc.subject | 雙硫鍵 | zh_TW |
| dc.subject | 牛胰臟去氧核糖核酸水解酶 | zh_TW |
| dc.subject | 蛋白質摺疊 | zh_TW |
| dc.subject | bovine pancreatic DNase | en |
| dc.subject | protein folding | en |
| dc.subject | disulfide | en |
| dc.title | 牛胰臟去氧核糖核酸水解酶中雙硫鍵之生物功能及其N與C端片段參與活性蛋白摺疊之重要性 | zh_TW |
| dc.title | Biological Functions of the Disulfides in Bovine Pancreatic Deoxyribonuclease and the Involvement of its N- and C-Terminal Fragments in Active Protein Folding | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 魏耀揮,張固剛,袁小琀,莊榮輝,張明富,張智芬,林榮耀 | |
| dc.subject.keyword | 牛胰臟去氧核糖核酸水解酶,蛋白質摺疊,雙硫鍵, | zh_TW |
| dc.subject.keyword | protein folding,bovine pancreatic DNase,disulfide, | en |
| dc.relation.page | 158 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2004-10-05 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-93-1.pdf 未授權公開取用 | 1.56 MB | Adobe PDF |
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