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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 廖大修(Ta-Hsiu Liao),呂紹俊(Shao-Chun Lu) | |
| dc.contributor.author | Ru-Ting Huang | en |
| dc.contributor.author | 黃如婷 | zh_TW |
| dc.date.accessioned | 2021-06-15T00:14:45Z | - |
| dc.date.available | 2009-09-15 | |
| dc.date.copyright | 2009-09-15 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-06-24 | |
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J Biol Chem 234, 3003-3006 8. Liao, T. H., Liao, W. C., Chang, H. C. and Lu, K. S. (1989) Deoxyribonuclease II purified from the isolated lysosomes of porcine spleen and from porcine liver homogenates. Comparison with deoxyribonuclease II purified from porcine spleen homogenates. Biochim Biophys Acta 1007, 15-22 9. Schafer, P., Cymerman, I. A., Bujnicki, J. M. and Meiss, G. (2007) Human lysosomal DNase IIalpha contains two requisite PLD-signature (HxK) motifs: evidence for a pseudodimeric structure of the active enzyme species. Protein Sci 16, 82-91 10. Odaka, C. and Mizuochi, T. (1999) Role of macrophage lysosomal enzymes in the degradation of nucleosomes of apoptotic cells. J Immunol 163, 5346-5352 11. McIlroy, D., Tanaka, M., Sakahira, H., Fukuyama, H., Suzuki, M., Yamamura, K., Ohsawa, Y., Uchiyama, Y. and Nagata, S. (2000) An auxiliary mode of apoptotic DNA fragmentation provided by phagocytes. Genes Dev 14, 549-558 12. Barry, M. A. and Eastman, A. (1993) Identification of deoxyribonuclease II as an endonuclease involved in apoptosis. Arch Biochem Biophys 300, 440-450 13. Yasuda, T., Takeshita, H., Iida, R., Nakajima, T., Hosomi, O., Nakashima, Y. and Kishi, K. (1998) Molecular cloning of the cDNA encoding human deoxyribonuclease II. J Biol Chem 273, 2610-2616 14. Krieser, R. J. and Eastman, A. (1998) The cloning and expression of human deoxyribonuclease II. A possible role in apoptosis. J Biol Che. 273, 30909-30914 15. Chou, S. F., Chen, H. L. and Lu, S. C. (2002) Up-regulation of human deoxyribonuclease II gene expression during myelomonocytic differentiation of HL-60 and THP-1 cells. Biochem Biophys Res commun 296, 48-53 16. Kawane, K., Fukuyama, H., Kondoh, G., Takeda, J., Ohsawa, Y., Uchiyama, Y. and Nagata, S. (2001) Requirement of DNase II for definitive erythropoiesis in the mouse fetal liver. Science 292, 1546-1549 17. Oshima, R. G. and Price, P. A. 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A procedure for the preparation of acid deoxyribonuclease and other acid hydrolases. Biochim Biophys Acta 129, 1-11 24. MacLea, K. S., Krieser, R. J. and Eastman, A. (2003) Structural requirements of human DNase II alpha for formation of the active enzyme: the role of the signal peptide, N-glycosylation, and disulphide bridging. Biochem J 371, 867-876 25. Mordier, S. B., Bechet, D. M., Roux, M. P., Obled, A. and Ferrara, M. J. (1995) The structure of the bovine cathepsin B gene. Genetic variability in the 3' untranslated region. Eur J Biochem 229, 35-44 26. Beyer, B. M. and Dunn, B. M. (1996) Self-activation of recombinant human lysosomal procathepsin D at a newly engineered cleavage junction, 'short' pseudocathepsin D. J Biol Chem 271, 15590-15596 27. Guimaraes, M. J., Bazan, J. F., Castagnola, J., Diaz, S., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Varki, A. and Zlotnik, A. (1996) Molecular cloning and characterization of lysosomal sialic acid O-acetylesterase. J Biol Chem 271, 13697-13705 28. Liao, Y. F., Lal, A. and Moremen, K. W. (1996) Cloning, expression, purification, and characterization of the human broad specificity lysosomal acid alpha-mannosidase. J Biol Chem 271, 28348-28358 29. Ord, T., Adessi, C., Wang, L. and Freeze, H. H. (1997) The cysteine proteinase gene cprG in Dictyostelium discoideum has a serine-rich domain that contains GlcNAc-1-P. Arch Biochem Biophys 339, 64-72 30. Merril, C. R., Goldman, D., Sedman, S. A. and Ebert, M. H. (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211, 1437-1438 31. Seglen, P. O. (1983) Inhibitors of lysosomal function. Methods Enzymol 96, 737-764 32. Blok, J., Mulder-Stapel, A. A., Ginsel, L. A. and Daems, W. T. (1981) The effect of chloroquine on lysosomal function and cell-coat glycoprotein transport in the absorptive cells of cultured human small-intestinal tissue. Cell Tissue Res 218, 227-251 33. Ohkuma, S. and Poole, B. (1978) Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci USA 75, 3327-3331 34. MacLea, K. S., Krieser, R. J. and Eastman, A. (2002) Revised structure of the active form of human deoxyribonuclease IIalpha. Biochem Biophys Res commun 292, 415-421 35. Takeshita, H., Yasuda, T., Iida, R., Nakajima, T., Hosomi, O., Nakashima, Y., Mori, S., Nomoto, H. and Kishi, K. (1998) Identification of the three non-identical subunits constituting human deoxyribonuclease II. FEBS Lett 40, 239-242 36. Yasuda, T., Takeshita, H., Iida, R., Nakajima, T., Hosomi, O., Nakashima, Y., Mori, S. and Kishi, K. (1999) Structural requirements of a human deoxyribonuclease II for the development of the active enzyme form, revealed by site-directed mutagenesis. Biochem Biophys Res commun 256, 591-594 37. Hasilik, A., von Figura, K., Conzelmann, E., Nehrkorn, H. and Sandhoff, K. (1982) Lysosomal enzyme precursors in human fibroblasts. Activation of cathepsin D precursor in vitro and activity of beta-hexosaminidase A precursor towards ganglioside GM2. Eur J Biochem 125, 317-321 38. Mahuran, D. J., Neote, K., Klavins, M. H., Leung, A. and Gravel, R. A. (1988) Proteolytic processing of pro-alpha and pro-beta precursors from human beta hexosaminidase. Generation of the mature alpha and betaabetab subunits. J Biol Chem 263, 4612-4618 39. Benes, P., Vetvicka, V. and Fusek, M. (2008) Cathepsin D-Many functions of one aspartic protease. Crit Rev Oncol Hematol 68, 12-28 40. Shacka, J. J. and Roth, K. A. (2007) Cathepsin D deficiency and NCL/Batten disease: there's more to death than apoptosis. Autophagy. 3, 474-476 41. Hasilik, A. and Neufeld, E. F. (1980) Biosynthesis of lysosomal enzymes in fibroblasts. Synthesis as precursors of higher molecular weight. J Biol Chem 255, 4937-4945 42. Gieselmann, V., Pohlmann, R., Hasilik, A. and Von Figura, K. (1983) Biosynthesis and transport of cathepsin D in cultured human fibroblasts. J Cell Biol 97, 1-5 43. Conner, G. E. (1989) Isolation of procathepsin D from mature cathepsin D by pepstatin affinity chromatography. Autocatalytic proteolysis of the zymogen form of the enzyme. Biochem J 263, 601-604 44. Green, G. D. and Shaw, E. (1981) Peptidyl diazomethyl ketones are specific inactivators of thiol proteinases. J Biol Chem 256, 1923-1928 45. Hentze, M., Hasilik, A. and von Figura, K. (1984) Enhanced degradation of cathepsin D synthesized in the presence of the threonine analog beta-hydroxynorvaline. Arch Biochem Biophys 230, 375-382 46. Nishimura, Y., Higaki, M. and Kato, K. (1987) Identification of a precursor form of cathepsin D in microsomal lumen: characterization of enzymatic activation and proteolytic. Biochem Biophys Res commun 148, 335-343 47. Cymerman, I. A., Meiss, G. and Bujnicki, J. M. (2005) DNase II is a member of the phospholipase D superfamily. Bioinformatics 21, 3959-3962 48. Huang, P. T., Chen, T. Y., Tseng, L. J., Lou, K. L., Liou, H. H., Lin, T. B., Spatz, H. C. and Shiau, Y. Y. (2002) Structural influence of hanatoxin binding on the carboxyl terminus of S3 segment in voltage-gated K(+)-channel Kv2.1. Receptors channels 8, 79-85 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41248 | - |
| dc.description.abstract | 由豬脾臟純化而得的乙型去氧核糖核酸酶 (porcine spleen DNase II)，簡稱為pDNase II，為一酸性DNA水解酶，具有α1、β及α2 三個包含於同一cDNA之次單元體，連接α1、β與β、α2間的連接肽鏈可能是藉轉譯後修飾作用而切除方形成pDNase II之三次元體結構。為探討轉譯後加工切除與pDNase II間的關聯性，我們利用暫時性轉染將載有pDNase II cDNA的表現質體 (pDNaseII)送入人類胚胎腎臟293T細胞 (HEK 293T cells)中表現重組pDNase II (rpDNase II)。在細胞萃取物中可見35、11.5以及46.5 kDa的蛋白質訊號，但在細胞培養液中僅見46.5 kDa的蛋白質訊號。在培養液中偵測到的46.5 kDa蛋白質藉由Cibacron Blue以及Mini-S管柱層析純化至均質並分析特徵。MALDI-TOF質譜儀和胺基酸定序的結果顯示此46.5 kDa蛋白質為具有單一多肽鏈的pDNase II。46.5 kDa的rpDNase II可於活性膠體染色分析中顯示酸性水解酶之酵素活性且比活性約為pDNase II的兩倍。轉染pcDNaseII的人類胚胎腎臟293T細胞經溶酶體抑制劑chloroquine處理後會造成46.5 kDa蛋白質訊號增加與35 kDa蛋白質訊號的減少。而以蛋白質生合成抑制劑cycloheximide處理人類胚胎腎臟293T細胞後，46.5 kDa的蛋白質訊號會逐漸消失且35kDa蛋白質訊號會增加。由以上結果可知rpDNase II的轉譯後加工切除發生於溶酶體中，且轉譯後加工切除並不參與pDNase II活化過程。
pDNase II具有六個N-醣基化位置，分別是Asn72、Asn88、Asn171、Asn214、Asn268與Asn292，我們利用定位點突變的方式將此六個天門冬醯胺 (asparagine)殘基分別突變成麩醯胺酸 (glutamine)，再將構築完成的突變株以暫時轉染方式送入人類胚胎腎臟293T細胞表現突變重組蛋白質，此六個突變蛋白質的酵素活性並不因點突變而有大幅減少，另外，經b-N-Acetylglucosaminidase H去除所攜帶醣基的rpDNase II，仍可在活性染色膠體上顯示活性。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2021-06-15T00:14:45Z (GMT). No. of bitstreams: 1 ntu-98-D89442002-1.pdf: 4597912 bytes, checksum: 175ed9986be9405a0ab113b3fcd94d00 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 第一章 研究背景、目的與實驗構想 1
第一節 前言 2 第二節 第二型（乙型）去氧核醣核酸水解酶 2 第三節 研究緣起 4 第二章 實驗材料與儀器 7 第一節 實驗材料 8 第二節 實驗儀器 9 第三章 實驗方法 11 第一節 以人類293T細胞表現rpDNase II 12 1. 細胞培養 12 2. 質體轉染 (Transfection) 12 3. 轉染後細胞培養液之收集及細胞萃取物之製備 13 4. 十二烷硫酸鈉聚丙烯醯胺板膠電泳法 (SDS-PAGE) 13 (1) 架設鑄膠裝置 13 (2) 配製12 %聚丙醯胺之分離膠體溶液 (separating gel) 13 (3) 配製 3 % 聚丙醯胺之焦集膠體溶液 (stacking gel） 14 (4) 樣品處理 14 (5) 電泳操作 14 5. 西方式墨點分析 (Western blot analysis) 14 (1) 半濕式蛋白質轉印法 14 (2) 免疫染色 15 6. DNase II活性測定 15 (1) Hyperchromicity 動力學法 15 (2) 洋菜培養基活性分析法(metachromatic agar-diffusion assay) 16 (3) DNase II膠體內活性呈色法 (Zymographic Analysis) 16 (i) 膠體製備 16 (ii) 活性呈色 16 第二節 rpDNase II之純化分離與特徵分析 16 1. 以Cibacron Blue 3-GA管柱進行rpDNaseII之分離 16 2. 以Mini-S管柱進行rpDNaseII之分離 17 3. 銀染色法 (silver staining) 17 4. rpDNaseII之質量測定 18 5. rpDNaseII之N-端胺基酸定序 18 第三節 rpDNase II於溶酶體內之加工切割作用與轉譯後切割 產物之相對穩定度 18 1. 以chloroquine抑制溶酶體內rpDNase II之轉譯加工切 割作用 18 2. 蛋白質合成抑制劑cycloheximide (CHX)實驗 19 第四節 以PCR定位突變方式建構N-醣基化突變質體及b-N-Acetylglucosaminidase H ( b-AGase H) 酵素反應 19 1. 利用QuikChangeTM Site-Directed Mutagenesis Kit method製備定點突變株 19 (1) PCR反應條件 19 (2) 洋菜膠體電泳 20 (3) Dpn I酵素反應 20 (4) 轉形作用 (Transformation) 20 (5) 質體 DNA小量抽取 21 (6) 質體的大量抽取 21 (7) DNA濃度測定法 22 2. 利用重疊延伸法 (Overlapping extension method)製備定點 突變株 23 (1) 第一次PCR 23 (2) 酒精沈澱PCR產物 24 (3) 融合反應 24 (4) 第二次PCR 24 (5) 限制酶水解反應 25 (6) DNA回收 25 (7) 接合反應 26 3. b-AGase H酵素反應 26 第四章 實驗結果 27 第一節 以人類293T細胞表現rpDNase II 28 1. DNase II活性分析 28 2. 轉染後蛋白質表現 28 第二節 rpDNase II之純化分離與特徵分析 29 第三節 膠體內酸性DNase活性分析 30 第四節 rpDNase II於溶酶體內之加工切割作用 31 第五節 rpDNase II的轉譯後切割產物於溶酶體中的相對穩定 度 31 第六節 N-醣基化作用對pDNase II的重要性 32 1. b-AGase H酵素反應 32 2. 六個單一突變株N72Q、N88Q、N214Q、N268Q與 N292Q的構築 33 3. 六個單一突變株在細胞培養液中的表現 33 4. 四個雙重突變株N72/88Q、N171/214Q、N214/268Q 和N268/292Q的構築與在細胞培養液中的表現 34 5. 多重突變株的構築與在細胞培養液中的表現 35 第五章 討論 36 第一節 pDNase II的轉譯後切割加工發生於溶酶體中 37 第二節 pDNase II的轉譯後切割加工作用並非其催化活性 之必要因素 38 第三節 對於可能參與DNase II轉譯後加工作用之溶酶體蛋白 質水解酶的推測 39 第四節 N-醣基化作用對pDNase II活性的重要性 40 第六章 圖表 42 第七章 參考文獻 79 | |
| dc.language.iso | zh-TW | |
| dc.subject | 去氧核醣核酸水解酶 | zh_TW |
| dc.subject | 轉譯後加工切割 | zh_TW |
| dc.subject | post-translational processing | en |
| dc.subject | Deoxyribonuclease II | en |
| dc.title | 溶酶體內轉譯後加工切割與N-醣基化作用對豬第二型去氧核醣核酸水解酶之重要性 | zh_TW |
| dc.title | The Significance of Lysosomal Post-translational Processing and N-glycosylation for Porcine Deoxyribonuclease II | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 林榮耀,周綠蘋,陳威戎 | |
| dc.subject.keyword | 去氧核醣核酸水解酶,轉譯後加工切割, | zh_TW |
| dc.subject.keyword | Deoxyribonuclease II,post-translational processing, | en |
| dc.relation.page | 90 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-06-24 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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