本研究利用SP Sepharose管柱層析，經過一個步驟即可將創傷弧菌核酸分解酵素（Vvn）由細胞周質萃取液中純化出來。純化的Vvn在reducing和non-reducing SDS-PAGE中具有不同的泳動性，因此推測雙硫鍵可能和Vvn之三級結構的穩定有關。Vvn具有任意切割單股和雙股的DNA以及RNA的核酸內切的活性。Vvn的最適pH值為pH 8.0 – pH 10.0；其DNase與RNase活性的最適溫度分別為40 oC - 60 oC與40 oC – 50 oC。EDTA和EGTA會抑制Vvn的活性，顯示其為metalloenzyme；此外，Vvn的DNase及RNase活性對二價金屬離子的需求不同。Vvn的化學修飾研究顯示lysine、arginine、tryptophan 以及含carboxylate group的胺基酸可能和Vvn的催化活性有關，而EDC修飾carboxylate group後對Vvn之DNase及RNase活性的抑制程度不同。受質DNA和RNA可以保護PLP、PGO、NBS及EDC （分別修飾lysine、arginine、tryptophan及carboxylate group）對Vvn之DNase及RNase活性的抑制作用。Mg2+對PLP和PGO的抑制作用無保護的能力，然而Mg2+可以保護NBS和EDC對Vvn之DNase活性的抑制作用但對Vvn之RNase活性的抑制作用則無影響。綜合本研究的結果，推論Vvn可能具有兩個活性中心，分別負責DNase及RNase的活性，但其活性中心有部份重疊的情形。

The periplasmic nuclease of Vibrio vulnificus, Vvn, has been purified to homogeneity by a one step purification procedure using chromatography on a SP Sepharose column. The purified enzyme showed different mobilities on reducing and non-reducing SDS-PAGE, suggesting that disulfide bonds are involved in the maintenance of a stable tertiary conformation of the protein. Vvn randomly cleaved single and double stranded DNA and RNA, and possessed endonucleolytic activity. The enzyme exhibited an optimal activity between pH 8.0 and pH 10.0, and the optimal temperatures for the DNase and RNase activity were 40 oC – 60 oC and 40 oC – 50 oC, respectively. The enzymatic activity was inhibited by EDTA and EGTA, indicating that Vvn was a metalloenzyme. The DNase and RNase activity of Vvn had different requirements for divalent cations. Chemical modification studies on Vvn revealed the involvement of lysine, arginine, tryptophan and carboxylate residues in the catalytic activity of the enzyme. The extents of inactivation of the DNase and RNase activity of Vvn by modification of the carboxylate group with EDC were different. Substrate DNA and RNA protected the DNase and RNase activity of Vvn from inactivation by PLP, PGO, NBS and EDC which modified lysine, arginine, tryptophan and the carboxylate group. Mg2+ could not protect the DNase and RNase activity of Vvn against the inactivation by PLP and PGO. Whereas Mg2+ protection was observed in NBS- and EDC-mediated inactivation of the DNase but not the RNase activity of Vvn . From these results, it is postulate that there may be two distinct but overlapping active sites, for the DNase and RNase activity, respectively.

緒論 …………………………………………………………………… 1
研究方法 ……………………………………………………………… 5
1. 菌種培養 ……………………………………………………… 5
2. Vvn的純化 …………………………………………………… 5
2.1. 細胞周質的萃取 ……………………………………… 6
2.2. 陽離子交換層析法 …………………………………… 6
3. 蛋白質膠體電泳法 (SDS-PAGE) …………………………… 7
4. 蛋白質濃度測定 ……………………………………………… 8
5. 受質專一性的分析 …………………………………………… 8
6. 核酸分解活性的分析 ………………………………………… 9
6.1. DNase活性分析 ………………………………………… 9
6.2. RNase活性分析 ………………………………………… 9
7. 化學修飾 ……………………………………………………… 10
7.1. DEPC的修飾作用 ……………………………………… 10
7.2. PMSF的修飾作用 ……………………………………… 11
7.3. PLP的修飾作用 ………………………………………… 11
7.4. PGO的修飾作用 ……………………………………… 12
7.5. NBS的修飾作用 ………………………………………… 12
7.6. EDC的修飾作用 ……………………………………… 13
7.7.受質及Mg2+的保護作用 ………………………………… 13
結果 …………………………………………………………………… 14
1. Vvn的純化 …………………………………………………… 14
2. Vvn的受質專一性 …………………………………………… 14
3. pH值和反應溫度對Vvn活性的影響 …………………………… 15
4. Vvn對熱的安定性 …………………………………………………… 16
5. 二價金屬離子對Vvn活性的影響 ………………………………… 16
6. 化學修飾作用對Vvn活性的影響 ………………………………… 17
6.1. DEPC和PMSF對Vvn活性的影響 ………………………… 17
6.2. PLP對Vvn活性的影響 ……………………………………… 17
6.3. PGO對Vvn活性的影響 ……………………………………… 18
6.4. NBS對Vvn活性的影響 ……………………………………… 19
6.5. EDC對Vvn活性的影響 ……………………………………… 20
討論 …………………………………………………………………… 22
參考文獻 ……………………………………………………………… 30
表 ……………………………………………………………………… 36
圖 ……………………………………………………………………… 43


表目錄
表1、Vvn的純化 …………………………………………………… 36
表2、二價金屬離子對Vvn活性的影響 …………………………… 37
表3、不同的化學修飾劑對Vvn活性的影響 ……………………… 38
表4、受質及Mg2+對PLP抑制作用的影響 …………………………… 39
表5、受質及Mg2+對PGO抑制作用的影響 …………………………… 40
表6、受質及Mg2+對NBS抑制作用的影響 …………………………… 41
表7、受質及Mg2+對EDC抑制作用的影響 …………………………… 42

圖目錄
圖1、Vvn、Vibrio cholerae Dns、Escherichia coli EndoI、Erwinia chrysanthemi NucM以及Aeromonas hydrophila Dns 的氨基
酸序列比對結果 ……………………………………………………… 43
圖2、SP Sepharose管柱層析之溶離圖譜 …………………………… 44
圖3、利用Reducing 和 non-reducing SDS-PAGE分析純化的Vvn 45
圖4、Vvn 分解plasmid DNA的情形 ……………………………… 46
圖5、Vvn分解chromosomal DNA的情形 ………………………… 47
圖6、Vvn分解 RNA的情形 ……………………………………… 48
圖7、Vvn 分解single-stranded DNA的情形 ……………………… 49
圖8、pH值對Vvn之DNase活性與RNase活性的影響 ………… 50
圖9、反應溫度對Vvn之DNase活性與RNase活性的影響 ……… 51
圖10、Vvn對熱的安定性 …………………………………………… 52
圖11、PLP對Vvn的抑制作用 ……………………………………… 53
圖12、PLP濃度對Vvn活性的影響 ………………………………… 54
圖13、PGO對Vvn的抑制作用 …………………………………… 55
圖14、PGO濃度對Vvn活性的影響 ……………………………… 56
圖15、NBS對Vvn的抑制作用 …………………………………… 57
圖16、Tryptophan修飾個數與Vvn活性的關係 …………………… 58
圖17、NBS濃度對Vvn活性的影響 ……………………………… 59
圖18、EDC對Vvn的抑制作用 …………………………………… 60
圖19、EDC濃度對Vvn活性的影響 ……………………………… 61

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