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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.author | 陳世峰 | zh_TW |
| dc.date.accessioned | 2021-07-01T08:12:05Z | - |
| dc.date.available | 2021-07-01T08:12:05Z | - |
| dc.date.issued | 2001 | |
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(2000) Leucaena Leucocephala serine proteinase inhibitor: Primary structure and action on blood coagulation, kinin release and rat paw edema. Biochimica et Biophysica Acta 1477:64-74 Oliveira, E. P., and Tanizaki, M. M. (1992) Effect of a proteinase inhibitor from the plasma of Bothrops jararaca of coagulant and mytotoxic activities of bothrops venomes. Toxicon30: 123-128 Pearce, G., Ryan, C. A., and Lijiegren, D. (1988) Proteinase inhibitors I and II infruit of wild tomato species: Transient components of a mechanism for defense and seed dispersal. Planta 175:527-531. Peitsch, M. C. (1996) ProMod and Swiss-Model: Internet-based tools for automated comparative protein modeling. Biochem. Soc. Trans. 24:274-279. Peitsch, M. C. (1995) Protein modeling by E-mail. Bio/Technology 13: 658-660. Plubkett, G., and Ryan, C. A. (1980) Reduction and carboxyamidomethylation of the single disulfide bond of proteinase inhibitor I from potato tubers. .J Biol. Chem. 255:2752-2755. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75174 | - |
| dc.description.abstract | SPLTI (sweet potato leaf trypsin inhibitor)為甘藷葉部經乾旱誘導產生的蛋白質，屬於Potato I family，經過初步分析胺基酸一級排列以及三級結構模擬，比對得到活性區的膠基酸為-R-E-，在Potato I family中較為特殊。SPLTI經由電泳分析得到的分子量為14kDa，為預估分子量的2倍；結構模擬的結果顯示SPLTI並不容易形成分子內雙硫鍵。因而本論文希望能證實-R-E-和TI (trypsin inhibitor)活性的關係以及SPLTI是否以雙硫鍵形成雙元體。於是進一步以定點突變，分析R46M、E47K、C42S和TI活性的關係，R46M與E47K突變蛋白喪失TI活性，C42S仍保有TI活性。接著以β-mercaptoethanol與dithiothreitol處理SPLTI，得到單元體蛋白質。C42S與C59S突變蛋白的分析，證實了雙元體是由此二胺基酸形成的雙硫鍵造成的。此外，也測試了SPLTI以及R46kFE47D雙突變蛋白對於胰凝乳蛋白脢及枯草桿菌蛋白脢的抑制效果，結果SPLTI以及R46M以及R46kFE47D突變蛋白對此二蛋白脢並沒有抑制作用。在酵素-抑制因數複合體的動力學研究，得到SPLTI對於胰蛋白脢的平衡解離常數Ki為0.81±0.24nM; C42S突變蛋白對於胰蛋白脢的Ki=1.73±0.26nM及C59S突變蛋白對於胰蛋白脢的Ki=1.64±0.24nM。最後則對雙元體形成的重要性，與抑制效果的專一性提出合理的解釋。 | zh_TW |
| dc.description.abstract | SPLTI (sweet potato leaf trypsin inhibitor) was drought-induced in sweet potato leaf. The previous study of SPLTI found that it belongs to Potato I family. It is different since the reactive sites of SPLTI might be -R-E- which is obtained by primary sequence alignment and three dimensional (3-D) structural modeling. The molecular weight of SPLTI was l4kDa when analysis by SDSPAGE. It is twice of the molecular weight of 7kDa deduced from amino acid sequence. No apparent disulfide bond was found through molecular modeling analysis. The objectives of the thesis were (1) to demonstrate the relationship of TI (trypsin inhibitor) activity and the reactive site, -R-E-; (2) to study whether the inter-molecular disulfide bonds contribute to dimerization of SPLTI. When using site-directed mutagenesis as the tool, it is proved that R46M and E47K mutated protein lost the TI activity, while C42S did not. The protomer of SPLTI can be separated when treated with β-mercaptoethanol and dithiothreitol, respectively. The contribution of disulfide bond to dimerization of SPLTI was demonstrated by C42S and C59S mutated protein as well. The SPLTI and R46M/E47D double mutated protein did not show inhibitory activity to chymotrypsin and subtilisin. The equilibrium dissociation constant (K1) of SPLTI to trypsin was 0.81±0.24 nM, C42S mutated protein to trypsin was 1.73±0.26nM, and C59S mutated protein to trypsin was 1.64±0.24nM. The importance of dimerization and the specific inhibitory activity of SPLTI were discussed. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-01T08:12:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2001 | en |
| dc.description.tableofcontents | 中文摘要…………………………Ⅰ 英文摘要…………………………Ⅱ 縮寫列表…………………………Ⅲ 胺基酸縮寫對照表…………………………Ⅳ 第一章 前言…………………………1 蛋白脢抑制因數…………………………1 蛋白脢抑制因數之分類…………………………2 PI-I蛋白質之簡介…………………………3 PI-I蛋白質在植物體扮演的角色…………………………3 PI-I蛋白質的分子結構…………………………4 PI-I蛋白質活性區的比較…………………………5 甘藷葉部胰蛋白脢抑制因數…………………………6 結構模擬和定點突變…………………………7 本論文之研究目的…………………………8 第二章 材料與方法…………………………9 實驗材料…………………………9 實驗方法…………………………9 1．勝任細胞的製備…………………………9 2．DNA聚合脢連鎖反應…………………………10 3．蛋白質3D結構模擬…………………………11 4．定點突變法…………………………11 5．限制脢酵素反應…………………………12 6．接合反應…………………………13 7．將接合DNA轉殖至大腸桿菌…………………………13 8．質體DNA的純化…………………………13 9．融合蛋白在大腸桿菌之最佳化表現…………………………14 10．大量表現融合蛋白…………………………15 11．pGEX-2T表現之融合蛋白的純化與切離…………………………15 12．pGEX-6P表現之融合蛋白的純化與切離…………………………17 13．Glutathion Sepharose 4B膠體的再生…………………………18 14．蛋白質定量分析…………………………18 15．SDS(sodium dodecyl sulfate)膠片的製備與蛋白質電泳分析………………19 16．Tricine膠片的製備及蛋白質電泳分析…………………………20 17．膠體中胰蛋白脢抑制因數活性分析…………………………21 18．免疫轉印法…………………………22 19．蛋白脢抑制因數的活性測定…………………………24 20．胰蛋白脢親和力Km值的測定…………………………25 21．胰蛋白騰抑制因數平衡解離常數之測定…………………………26 第三章 結果…………………………28 1．蛋白質立體結構之預測與相似性模擬…………………………28 2．構築pGEX-2T/SPLTI，pGEX-6P/SPLTI重組質體…………………………33 3．大腸桿菌表現GST-LTI融合蛋白質…………………………33 4．利用定點突變建立SPLTI突變株…………………………37 5．SPLTI突變蛋白質的表現…………………………37 6．SPLTI重組蛋白之單元體與雙元體之分析…………………………39 7．蛋白脢抑制因數的活性測定…………………………44 8．胰蛋白脢抑制因數平衡解離常數之測定…………………………48 第四章 討論…………………………51 參考文獻…………………………55 附圖…………………………60 | |
| dc.language.iso | zh-TW | |
| dc.title | 甘藷葉部PI-I胰蛋白?仰制因數活性區及雙元體構造之探討 | zh_TW |
| dc.title | Survey on the Reactive Site and Dimerization of Proteinase Inhibitor-I from Sweet Potato | en |
| dc.date.schoolyear | 89-2 | |
| dc.description.degree | 碩士 | |
| dc.relation.page | 65 | |
| dc.rights.note | 未授權 | |
| dc.contributor.author-dept | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| 顯示於系所單位： | 植物科學研究所 | |
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