臺灣博碩士論文加值系統

由牛、羊、豬、兔子、人類、雞及魚等脊椎動物中純化獲得的去氧核醣核酸(Deoxyribonuclease, DNase)，主要存在於胰臟、腮腺等消化腺體內，此酵素只能以DNA作為受質。另一方面，由非脊椎動物中，如蝦子肝胰臟中純化所得的酵素，則可水解DNA或RNA，也就是同一蛋白質具有DNase及RNase兩種活性，稱其為核酸(Nuclease)。為了探討核酸由較低等生物至哺乳類動物演化上的差異，在此篇論文中我們以軟體動物門之阿根廷魷魚(Illex argentinus)作為實驗材料，純化其肝胰臟內核酸。
以30-80 %硫酸銨沉澱法所得到的粗抽液，經過Macro-Prep High Q、Phenyl Sepharose、Sephadex G-100 column等管柱層析法純化後，以SDS-PAGE證明已達到均質化，同時以去氧核醣核活性染色法，證明此酵素的分子量為45 kDa，且為單元體。魷魚中純化所得的核酸比活性為4800 units/mg，最佳活性的酸鹼值為7.0，在10 mM CaCl2、20 mM MgCl2同時存在下活性最高。以等電點焦集電泳法證明此酵素的等電點值約為3.5，且無同功(isoforms)的存在。與牛胰臟去氧核醣核酸相似，兩者皆於高pH值下較穩定(pH6.5~7.5)。以醣類親和性管柱層析法分析，證明魷魚肝臟內核酸並不會吸附於Con A管柱上，故並不像牛胰臟去氧核醣核酸一樣為醣蛋白，反而與蝦子肝胰臟核酸的性質相同。另一點與蝦子核酸相似處為SDS-PAGE分析時，配合不同的樣本處理方式，即是否加入2-Mercaptoethanol及Sodium Dodecyl Sulfate，配合加熱與否，可使魷魚肝臟核酸在SDS-PAGE上呈現多形態，分別為21、45 kDa及兩者間smear部分；若SDS存在下加熱，可形成45 kDa單一色帶。由溫度穩定性的
實驗得知魷魚肝臟核酸的Tm值約為40 ℃。
胺基酸組成分析結果顯示，魷魚肝臟核酸具有相當高量的Aspartate及Glutamate，此點與蝦子核酸相同，可能為造成其低等電點值的原因。具有十二個半胱胺酸(Cysteine)，與蝦子核酸十一個相似，遠較牛胰臟去氧核醣核酸四個為多；可能可形成六對雙硫鍵，使整個蛋白質的構形較為緊密，造成SDS-PAGE分析時，因SDS不易插入此酵素內部，造成變性(denaturation)不完全，故在SDS-PAGE上呈現多形態。以RNase活性染色法證明魷魚肝臟核酸也具有RNase的活性，故此酵素亦為一核酸與蝦子核酸之性質相同。利用70 % formic acid在37 ℃水浴下加熱48小時，可以將魷魚核酸切成兩個片段，以SDS-PAGE分析，分子量分別為14、31 kDa。31 kDa胺基酸序列分析的結果為：(Asp)Pro-Ser-Tyr-Pro-Ser-Ile-Lys-Thr，14 kDa胺基酸序列分析的結果為：N terminal block，推測此段為原先蛋白質的N端。
由以上結果我們可以得知在低等生物中，如節肢動物門之蝦子、軟體動物門之魷魚體內均為核酸，而較高等生物中均只含有只能水解DNA的去氧核醣核酸，推測在演化過程中乃由具有兩種功能的核酸(DNase、RNase activity)演變至只能分別水解DNA的去氧核醣核酸及水解RNA的核醣核酸。

Mammalian pancreatic deoxyribonuclease (DNase) is highly specific for DNA as substrate. Shrimp hepatopancreatic nuclease, on the other hand, is a nuclease. Nucleases, are enzymes capable of hydrolyzing both DNA and RNA, in a broad sense, should include DNase, RNase and the sugar-non-specific nuclease. To reveal whether or not the mammalian DNases are evolved from the shrimp nuclease, herein we described the investigation of yet another nuclease from squid hepatopancreas.
The hepatopancreatic tissue of squid from Argentina (Illex argentinus) was homogenized and the extract was subjected to ammonium sulfate precipitation followed by chromatography on Macro-Prep High Q, Phenyl Sepharose CL-4B, and Sephadex G-100 columns. The enzyme thus purified was homogenous as evidenced by a single band of Mr= 45 kDa on SDS-PAGE with silver-stain and DNase activity staining showed that the band coincided with DNase activity.
Squid nuclease has a specific activity of 4.8x103 units/mg protein, a pH-activity optimum of 7.0 and the best metal ion requirements of 20 mM MgCl2 plus 10 mM CaCl2. Squid nuclease was a very acidic protein (pI= 3.5), but was more stable at higher pH’s. Sugar-affinity chromatography revealed that squid nuclease was not a glycoprotein, a nature similar to shrimp nuclease. Thermal stability experiments showed that squid nuclease had a Tm of about 40 ℃. Like shrimp nuclease, squid nuclease showed at least three forms on SDS-PAGE, depending on the sample treatments, such as in the presence of sodium dodecyl sulfate and β-mercaptoethanol and with or without heating at 95 ºC. The amino acid composition of squid nuclease showed a high content of aspartate and glutamate, in agreement
with the acidic nature of the enzyme. Squid nuclease has an intrinsic RNase activity as analyzed by the RNase zymogram method. Thus, like the shrimp enzyme, squid DNase is a nuclease. The N-terminal amino acid residue was blocked and could not be determinated on a protein sequencer. Currently, we are trying to determine the cDNA sequence for squid nuclease with primers designed from the sequences of the peptides derived from formic acid cleavage or trypsin digestion.

誌謝I
目錄II
表目錄VII
圖目錄VIII
中文摘要IX
英文摘要XI
縮寫表XII
壹、緒論 1
一、去氧核醣核酸簡介 2
二、牛胰臟去氧核醣核酸 3
三、DNase I的作用機轉 6
四、阿根廷魷魚的簡介 9
貳、實驗藥品與儀器 10
一、實驗材料與藥品 11
二、實驗儀器 12
參、實驗方法 14
第一節 魷魚肝胰臟核酸水解的純化 15
一、樣本來源及保存 15
二、粗抽 15
三、硫酸銨沉澱法 15
四、透析 16
五、濃縮 16
六、陰離子交換樹脂層析法 16
七、疏水性管柱層析法 17
八、膠體過濾管柱層析法 18
九、蛋白定量 18
十、去氧核醣核酸的活性測定(Agar diffusion method) 18
十一、去氧核醣核酸的活性測定 19
十二、微透析 20
十三、膠體過濾管柱層析法 20
十四、真空幫浦濃縮 20
十五、洋膠盤製法 21
第二節 魷魚肝胰臟核酸水解之生化特性探討 22
一、十二烷硫酸鈉聚丙烯醯胺板膠電泳法 22
二、銀染色法 25
三、去氧核醣核酸活性染色法 26
四、Coomassie blue染色法 28
五、等電點焦集電泳法 30
六、PVDF membrane transfer 31
七、刀豆素親合性管柱層析法 32
八、pH值對魷魚肝臟中核酸水解穩定性的影響 32
九、pH值對魷魚核酸水解活性的影響 33
十、溫度對魷魚核酸水解活性的影響 33
十一、金屬離子對魷魚核酸水解活性的影響 34
十二、Multiple forms of squid nuclease on SDS-PAGE 35
十三、RNase活性染色法 36
十四、Cleavage Asp-Pro bond with 70 % formic acid 37
十五、Trypsin digestion 39
十六、酵素動力學測定 39
第三節 胺基酸分析 39
一、一般胺基酸分析 39
二、半胱胺酸分析(Cysteic acid) 41
三、色胺酸分析(Tryptophan) 41
第四節 cDNA基因庫的建立 42
一、RNA萃取 42
二、cDNA基因庫之建 44
三、聚合連鎖反應 45
肆、實驗結果與討論 47
第一節 魷魚肝胰臟核酸水解的純化 48
一、硫酸銨沉澱法 48
二、管柱層析法 48
第二節 魷魚肝胰臟核酸水解之生化特性探討 51
一、分子量的決定 52
二、魷魚中核酸水解等電點的決定 53
三、刀豆素親合性管柱層析法 53
四、pH值對魷魚肝臟中核酸水解穩定性的影響 54
五、pH值對魷魚肝臟中核酸水解活性的影響 55
六、溫度對魷魚核酸水解活性的影響 56
七、金屬離子對魷魚核酸水解活性的影響 56
八、Multiple forms of squid nuclease on SDS-PAGE 57
九、RNase活性染色 60
十、Cleavage Asp-Pro bond with 70 % formic acid 61
十一、Trypsin digestion 62
十二、酵素動力學分析 62
第三節 胺基酸分析 63
伍、結論及未來展望 65
第一節 起源 66
第二節 魷魚肝臟核酸水解的純化與性質分析 66
第三節 魷魚肝胰臟核酸水解的研究之未來展望 67
陸、實驗圖表 70
柒、參考文獻 95
捌、附錄 103

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劉明柏(1993)
(I) 牛尿液與牛胰臟去氧核醣核酸分子量差異之研究
(II) 去氧核醣核酸經牛尿中某因子修剪仍具活性的探討
國立臺灣大學醫學院生化學研究所碩士論文
蕭懿民(1994)
吳郭魚去氧核醣核酸之純化及其性質與胺基酸序列分析
國立臺灣大學醫學院生化學研究所碩士論文
陳瓊如(1999)
魷魚肝臟組織核酸水解之純化及其性質分析
國立臺灣大學醫學院生化學研究所碩士論文
王政清(1999)
豬脾臟乙型去氧核醣核酸水解之共價鍵結構及其 cDNA之選殖及表現
國立臺灣大學醫學院生化學研究所博士論文
椎野孝雄(昭和44年)
水產無脊椎動物學