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研究生:林錦泉
研究生(外文):Ching-Chuan Lin
論文名稱:草菇甲型去氧核醣核酸之純化及酵素性質研究
論文名稱(外文):Purification and Characterization of mushroom DNase I
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:生化學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:118
中文關鍵詞:去氧核醣核酸草菇胰蛋白水解硫酸銨
外文關鍵詞:DNase Imushroom (Volvariella volvacea)trypsinammonium sulfate
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草菇粗抽液在80 %硫酸銨存在下,於4 oC下攪拌48小時後,所沈澱下來之DNase I活性可以增加至4倍。經過40 %硫酸銨回溶回收,通過Phenyl Sepharose、Sephadex G-100、Hydroxyapatite及Superose 12等四種管柱層析分離法將所要的蛋白純化,從SDS-PAGE上可看到分子量為66 kDa的單一蛋白色帶,證明此一蛋白已達均質化(homogeneous)。而利用分子篩濾法(gel filtration)可計算出其分子量約為71 kDa。從等電點焦集電泳法(IEF)得到草菇DNase I的等電點為4.56,且無同功存在。此外,草菇DNase I無法吸附在Con A管柱上,可知其不是醣蛋白。
純化後的草菇DNase I經轉染至PVDF膜上,經N端定序得知其N端被修飾而無法讀出訊號。而純化後的草菇DNase I經Sephadex G-25管柱去離子化後,發現蛋白被降解成分子量30 kDa及28 kDa的主要片段,其N端分別為TRVKGA及QLPPINSPKSNL。另外利用胰蛋白水解(trypsin)水解草菇DNase I,可得到一分子量為27 kDa的片段,其N端為YYQLPPINSPKSNLFIILNY。去離子化的草菇DNase I在室溫下48小時候,其活性完全喪失,然而在1.0 M硫酸銨或5 mM PMSF存在下,草菇DNase I仍然可以保持70%及40 %的活性。由加熱的實驗顯示不論在硫酸銨存在與否,草菇DNase I的Tm值皆為55 oC。由以上的實驗可知在高離子濃度時,蛋白水解的活性被抑制,因此草菇DNase I在1.0 M硫酸銨存在下較為穩定。
草菇DNase I活性最適pH值為7.0,此和牛胰臟DNase I相同,但只有Ca2+存在時,草菇DNase I亦有活性,此與牛胰臟DNase I不同。此外,草菇DNase I在Mg2+ 或Ca2+單一存在時,就具有一次水解DNA兩股的能力。而牛胰臟DNase I在Mg2+單一存在時,只能一次水解DNA的單股,而在Mg2+ 及Ca2+同時存在時,才具有一次水解DNA兩股的能力。
在未來除了對草菇DNase I做其功能及生化上的研究外,我們希望可以得到草菇DNase I的cDNA序列,並和其他物種做比較。

The DNase I activity was raised about 4-fold when the crude extract of mushroom (Volvariella volvacea) was left in 80% ammonium sulfate at 4 oC for 48 hours. The DNase I was then purified from the (NH4)2SO4 precipitate by chromatography on the Phenyl Sepharose, Sephadex G-100, hydroxyapatite and Superose 12 columns. The purified enzyme was homogeneous as evidenced by SDS-PAGE with Mr = 66 kDa and by gel filtration on Superose 12 with Mr = 71 kDa. By isoelectric focusing on thin-layer polyacrylamide gel, the enzyme exhibited a single band with an acidic pI value of 4.56. The mushroom DNase I is not a glycoprotein because it can not be bound to Con A Sepharose.
After SDS-PAGE, mushroom DNase I was blotted to a PVDF membrane and subjected to protein sequencing, the results showed that the N-terminus was blocked. When the purified enzyme was desalted on Sephadex G-25 column, it was gradually degraded to yield two major fragments, 30 kDa and 28 kDa. The protein sequencing showed that the N-termini of these two fragments were TRVKGA and QLPPINSPKSNL, respectively. The N-terminus of a fragment of the trypsin digested mushroom DNase I was YYQLPPINSPKSNLFIILNY. The activity of the desalted mushroom DNase I was completely lost after 60 hours at room temperature, but the enzyme with 1.0 M (NH4)2SO4 or 5 mM PMSF still kept 70% and 40% activity respectively under the same condition. The heat experiment showed that with and without ammonium sulfate, the inactivation melting temperature of mushroom DNase I was 55 oC. The experiments indicated that mushroom DNase I was stable in the presence of 1.0 M (NH4)2SO4, because the protease activities were inhibited under the high salt condition.
Mushroom DNase I had an optimal pH = 7.0, but unlike mammalian DNase I, it was active with Ca2+ alone. On the other hand, mushroom DNase I introduces double strand cutting in the presence of either Mg2+ or Ca2+. Bovine DNase I, on the other hand, introduces only single strand nicks in the presence of Mg2+, while with Mg2+ plus Ca2+ can bovine DNase I introduce double strand cutting.
In the future, we will sequence the cDNA of mushroom DNase I and compare the differences among species and relate them to biochemical functions.

目 錄
中文摘要………………………………………1
英文摘要………………………………………3
壹、縮寫……………………………….............5
貳、緒論………………………………………7
參、實驗材料與儀器.……………………….14
肆、實驗方法……………………………… 17
伍、結果…………………………………….49
陸、討論…………………………………….58
柒、圖表……………………………………..71
捌、參考文獻……………………………….103
玖、附錄…………………………………….110

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