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研究生:廖政榮
研究生(外文):Liao, Cheng Jung
論文名稱:探討鏈激脢N端胜太鏈在人類血纖維蛋白溶脢原構型活化上所扮演的角色
論文名稱(外文):studying the role of the amino-terminal sequence of streptokinase in the formational activation of human plasminogen
指導教授:吳華林
指導教授(外文):Wu, Hua-Lin
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
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:101
中文關鍵詞:鏈激脢人類血纖維蛋白溶脢原構型活化N端胜太鏈異白胺酸
外文關鍵詞:streptokinasehuman plasminogenconformaitonal activationN-terminal peptideisoleucine
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鏈激脢 ( streptokinase, SK ) 是人類血纖維蛋白溶脢原(human plasminogen, HPlg)的活化子,含有414個胺基酸,分子量約45kDa的單一 鏈,由β-溶血性鏈球菌所分泌的菌體外蛋白質。SK能與HPlg形成莫爾比1:1之活化複合體,此複合體能水解游離態HPlg中Arg561-Val562間的 鏈,而將游離態的HPlg活化為HPlm;新生的Val562 N端會與Asp740形成salt bridge,造成活化區構型的改變,使HPlm具有蛋白水解脢的活性,此過程類似胰蛋白脢原(trypsinogen)活化成胰蛋白脢(trypsin)。活化的HPlm能水解血纖維蛋白,造成血塊的溶解。
SK誘導HPlg出現”virgin enzyme”活性的過程,並不會水解Arg561-Val562間的 鏈。SK活化HPlg的機制目前尚未清楚,有一”binding activation”假說認為,當SK與HPlg結合時,SK的γ定義區能與HPlg的autolysis loop交互作用,造成Lys698構型的改變,使Lys698與Asp740產生salt bridge,進而促成活化區的暴露。然而另有研究顯示,SK的Ile1才是與Asp740形成salt bridge的重要胺基酸,當SK缺少了Ile1,HPlg的活化區便無法形成,且SK之N端 鏈能穩定活化複合體;後者被稱為molecular sexuality 假說。
為釐清SK Ile1及N端 鏈在HPlg活化中所扮演的角色。本實驗室構築且純化出SK重組蛋白SK (1-378)、SK (2-378)、SK (16-378)、SK (Gly-1-378)、SK (Ile-Tag-16-378)(N端胺基酸定序分析90%的SK (1-378)和SK (Ile-Tag-16-378)在Ile前另有Met)。比活性及HPlg活化試驗中,所有SK突變株均能如原型SK有效活化HPlg。酵素動力學試驗顯示,各突變株的kcat和Km值沒有明顯差異。當以莫爾濃度比SK:HPlg=6:1、 4:1、 2:1或1:1之SK誘導HPlg的醯胺水解活性(amidolytic activity)時,HPlg的醯胺水解活性的誘導會隨著SK濃度的增加而受到延滯,但是原型SK (native SK)和SK (1-378)則無此現象。4-methylumbelliferyl p-guanidinobenzoate(MUGB) titration實驗顯示:等莫爾濃度下,各突變株均能使HPlg產生活化區。以非連續法分析HPlg之virgin enzyme活性,發現除了原型SK及SK (1-378),當過量的SK越多時,virgin enzyme活性也會越晚出現。
以催化濃度的SK活化HPlg時,缺少了N端序列或以Gly遮蔽Ile1之N端amino group並不影響SK活化HPlg。然而以過量的SK誘導HPlg活化時,隨著N端胺基酸截短的越多,HPlg的活化會有越長的延滯期,而在SK (16-378)的N端接上T7∙Tag,能減少延滯期的時間,顯示SK的N端在過量SK誘導HPlg活化時才扮演重要的角色,但此一交互作用的專一性並不高,因為與SK(1-15)無同源性的序列T7∙Tag亦能部分取代SK (1-15)的功能。此外SK (1-378)在N端多了Met,但是其活化HPlg的表現仍與原型SK相似,而SK (Gly-1-378)卻有延滯期的出現,顯示第一胺基酸不一定得是Ile,Met也能取代Ile的角色,但是中性的Gly便無法取代其功能。由以上結果推論,SK的N端序列在誘導HPlg構形活化上並不扮演重要的角色,但以過量SK誘導HPlg構型活化時,SK的N端可能與μ-HPlg作用,保護活化區不受過量SK的干擾,進而加速HPlg的構型活化。

Streptokinase (SK) is a single-peptide secretory protein of 414 amino acid residues produced by various strains of β-hemolytic Streptococcus. The SK and human plasminogen (HPlg) can form an equimolar activator complex that catalyzes the cleavage of the Agr561-Val562 peptide bond of HPlg to human plasmin (HPlm); the newly formed N-terminus of Val562 is believed to insert inwardly to form a salt bridge with the carboxylate of Asp740 in a manner analogous to the activation of trypsinogen to trypsin. Plm is a potent protease that in turn catalyzes the hydrolysis of fibrin, which causes the dissolution of blood clot.
By an unclearly-identified mechanism, SK can activate HPlg protease activity without the proteolytic cleavage. The “binding activation” hypothesis suggests that the binding of SK γdomain to the autolysis loop region of HPlg may cause a conformation change of Lys698, resulting in the formation of the critical salt bridge with Asp740 and thereby the activation of HPlg catalytic apparatus. However, recent reports have proposed that the N-terminus of SK might directly insert into μPlg domain to form salt linkage with Asp740, which was called “molecular sexuality” hypothesis.
In order to examine the function of Ile 1 and the N-terminal peptide of SK in the conformational activation of HPlg, we designed various SK mutants, including SK (1-378), SK (2-378), SK (16-378), SK (Gly-1-378) (with Gly residure to block the N-terminus of Ile1), and SK (Ile-Tag-16-378) (with Ile residue in the N-terminus); N-terminal amino acid sequencing revealed that SK (1-378) and SK (Ile-Tag-16-378) contain 90% N-terminal Met. According to specific activity and HPlg activation assays, all SK mutants could activate HPlg as efficiently as native SK with a catalytic concentration of SK. The kcat and Km values among those mutants were also similar. However, when the amount of SK is more than HPlg, the appearance of amidolytic activity was delayed in all the SK mutants except native SK and SK (1-378). The active site titration of HPlg with 4-methylumbelliferyl p-guanidinobenzoate(MUGB) revealed that all mutants could generate an active site in HPlg in an equimolar mixture, and that without free N-terminus or the Ile1 residue of SK , the exposure of active site was significantly inhibited under excess SK condition. Discontinuous amidolytic activity assays indicated that deletion or capping in the SK N-terminus would cause the delay of exposure of active site in HPlg.
Ile1 of SK may not play an critical role in the activation of HPlg with catalytic amount of SK, because the N-terminal deletion and the N-terminal blocking with Gly did not affect the activity of SK mutants. However, in the activation of HPlg with excess SK, the N-terminal deletion and the N-termial blocking with Gly would cause the delay of the HPlg conformational activation, and adding T7∙Tag in the N-terminal of SK (16-378) could decrease this effect. It was auggested that SK N-terminal sequence may be important in the HPlg activation with excess SK, and that the interaction of SK (1-15) with HPlg would not be highly specific, because even the T7∙Tag could subtitude the function of SK (1-15). Bescides, althought SK (1-378) had initiation Met in the N-terminal, the behavior was similar as native SK, but SK (Gly-1-378) had delay time in induction of HPlg conformaitonal activation. This result implied that Ile1 may not be an critital residue for initiation activation of HPlg, but a hydrophobic residue such as Met could be in place of the function of Ile1.
To sum up those observations, Ile1 of SK would not be an significant residue in the activation of HPlg with catalytic amount of SK. But when SK is over than HPlg, the excess SK may influence the formation of the active SK-HPlg complex, and the N-terminal sequence of SK could prevent the interaction of excess to SK-HPlg complex, therefore promoting the formation of SK-HPlg virgin enzyme activity.

中文摘要………………………………………………………………... 1
英文摘要………………………………………………………………… 3
致謝……………………………………………………………………… 6
目錄……………………………………………………………………… 7
表、圖、附錄目錄……………………………………………………… 10
縮寫檢索表……………………………………………………………… 11
緒論……………………………………………………………………… 13
藥品及材料……………………………………………………………… 20
儀器……………………………………………………………………… 24
方法……………………………………………………………………… 26
1. DNA基本技術操作與菌種保存…………………………………... 26
1-1. 小量質體DNA抽取……………………………………… 26
1-2. 瓊脂膠電泳分析…………………………………………. 26
1-3. 以電泳法回收DNA……………………………………… 28
1-4. 限制脢切割………………………………………………. 29
1-5. 接合反應…………………………………………………. 30
1-6. 形質轉移…………………………………………………. 32
1-7. 核酸分析定序……………………………………………. 34
1-8. 長期菌種保存……………………………………………. 34
2. 突變鏈激脢重組基因的構築、選殖及pET蛋白表現系統的建立. 34
2-1. 突變鏈激脢(SK (1-378)、SK (2-378)和SK (16-378)重組基因的構築………………………………………………. 34
2-2. 突變鏈激脢(SK (Gly-1-378)和SK (Ile-Tag-16-378))重組基因的構築………………………………………………. 36
2-3. 突變鏈激脢重組基因的選殖……………………………. 37
2-4. pET表現系統的建立……………………………………... 38
3. 鏈激脢突變株蛋白的表現、純化及N端胺基酸序列分析……….. 39
4. 蛋白濃度測定…………………………………………………...….. 41
5. SDS-PAGE電泳分析………………………………………………. 42
6. 鏈激脢的比活性測定………………………………………………. 45
7. 以連續法分析催化濃度之鏈激脢活化人類血纖維蛋白溶脢原的活性………………………………………………………………... 46
8. 以二步驟法分析催化濃度之鏈激脢活化人類血纖維蛋白溶脢原的活性……………………………………………….…………….. 47
9. 鏈激脢活化人類血纖維蛋白溶脢原恆定狀態的動力常數分析…. 48
10. 以過量濃度SK誘導人類血纖維蛋白溶脢原之醯胺水解活性….. 49
11. 以非連續法分析鏈激脢與人類血纖維蛋白溶脢原複合形成”virgin enzyme”之醯胺水解活性………………………………. 50
12. 鏈激脢誘導人類血纖維蛋白溶脢原形成活化中心的測定…….… 51
13. 鏈激脢與微小型人類血纖維蛋白溶脢原之BS3交叉連結反應分析…. 52
結果.
1. 鏈激脢突變株基因的構築與選殖…………………………..…….. 54
2. 鏈激脢突變株蛋白的表現、純化與N端胺基酸定序分析………. 54
3. 鏈激脢突變株做為人類血纖維蛋白溶脢原活化因子活性分析結果…………………………………………………………………….. 55
4. 鏈激脢突變株誘導人類血纖維蛋白溶脢原形成”virgin enzyme”活性分析結果…………………………………………….……….. 56
討論…………………………………………………………….………... 59
參考資料……………………………………………………….………... 64
表………………………………………………………………………… 69
圖………………………………………………………………………… 72
附錄……………………………………………………………………… 97
自述……………………………………………………………………… 101

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