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研究生:許煥宗
研究生(外文):HUAN-TSUNG HSU
論文名稱:蝦肝胰臟去氧核醣核酸水解酶211位置組胺酸為其酵素活性區之研究
論文名稱(外文):Evidence for His211 as the Active Site of Shrimp Hepatopancreas Nuclease
指導教授:廖大修
指導教授(外文):TA-HSIU LIAO
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物化學暨分子生物學研究所
學門:生命科學學門
學類:生物化學學類
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
論文頁數:77
中文關鍵詞:核酸水解酶酵素活性區
外文關鍵詞:Nucleaseactive site
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核酸水解酶(Nuclease)為水解DNA及RNA的酵素,由牛胰臟中純化所的到的甲型去氧核醣核酸酶(Deoxyribonuclease I, DNase I)是目前為止被研究的最透徹的;由蝦肝胰臟中所純化得到的nuclease具有典型的DNase I特徵,但是其胺基酸序列卻與牛DNase I的序列大不相同。在經過與其他種類DNase I比較後,我們發現蝦nuclease與DNase I序列並沒有任何的相似性;但是,若將蝦nuclease的序列跟其他nucleases的序列對照的話,蝦nuclease的序列205-255位置與其他nucleases在酵素活性功能區存在著共有的序列。
相較於過去的實驗報告,本研究提高了蝦nuclease在純化過程的回收率,蝦肝胰臟組織與一次水以1:2的比例混合均質化後的粗抽液,經過DEAE-cellulose、Sephadex G-100、Phenyl-Sepharose、Hydroxyapatite等管柱層析法純化後,以SDS-PAGE證明已達均質化;將蝦nuclease與10 mM MgCl2 及5 mM iodoacetate在37 ℃下靜置48小時後,95%的酵素活性會被抑制,因此利用iodo[2-14C]acetate對活性區的胺基酸標定,經過尿素及β-MSH作用將蛋白打開成直鏈狀,再利用thermolysin切割,通過Superdex Peptide PE7.5/300管柱可得到三段帶有放射線的肽片段;胺基酸組成分析結果顯示,在鎂離子存在下,IAA會與211位置組胺酸及294位置甲硫胺酸反應,但是在沒有鎂離子存在下,294位置甲硫胺酸也會與IAA反應,且酵素活性下降了約30%;因此我們推論211位置的組胺酸是蝦nuclease的催化活性中心,而294位置甲硫胺酸在酵素催化反應上可能也扮演一重要的角色。

Nucleases, in a broad sense, are enzymes capable of hydrolyzing both DNA and RNA. Bovine pancreatic DNase I (EC 3.1.21.1) is the DNase I that has been studied most thoroughly. Although the enzymatic properties of shrimp nuclease are typical of bovine DNase I, its sequence is distinct from those of bovine DNase I and other vertebrates. The protein and cDNA sequences for shrimp nuclease have been determined. Surprisingly, the sequences exhibited no similarity to those of the bovine DNase-I-like proteins. However, in the motif of residues 205-255, a number of identical residues were found when aligned with the conserved active sites of several nucleases.
We improved the recovery of shrimp nuclease during purification. Briefly, the hepatopancreatic tissue of shrimp was homogenized with deionized H2O at the ratio of 1:2, followed by chromatography on the DEAE-cellulose, Sephadex G-100, Phenyl-Sepharose, and two consecutive hydroxyapatite columns. The enzyme was purified to homogeneity as evidenced by a single band of Mr 45 kDa on SDS-PAGE. Shrimp nuclease in a solution containing 10 mM MgCl2 and 5 mM iodoacetate at 37 ℃ was 95 % inactivated in 48 hours. For the identification of the probable active site residues, shrimp nuclease was first modified with iodo[2-14C]acetate. Three major radioactive peptides were then isolated by chromatography on a Superdex Peptide PE7.5/300 column from the thermolytic digest of the labelled enzyme. The identification of the radioactive peptides by amino acid composition analysis revealed that the radio-labelled residues were His211 and Met294 when modification was carried out in the presence of Mg2+. However, Met294 could still be labelled by IAA in the absence of Mg2+, and the activity decreased 30%. Therefore, His211 is considered to be the active site of shrimp nuclease, and Met294 may also be involved in other unknown functions of catalysis.

中文摘要…………………………… 2
英文摘要…………………………… 4
壹、縮寫…………………………… 6
貳、緒論…………………………… 7
參、實驗材料與儀器……………… 14
肆、實驗方法……………………… 16
伍、結果…………………………… 31
陸、討論…………………………… 40
柒、圖表…………………………… 45
捌、參考文獻……………………… 74

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