臺灣博碩士論文加值系統

摘要

本論文由聚合酶聯鎖反應，選殖出樟芝子實體的thioredoxin reductase (簡稱TR) cDNA。全長為 1273 bp，轉譯區為1032 bp，譯出343個胺基酸。分析其序列上各domain 的分布：N端FAD binding domain序列為位於第29胺基酸起的GSGPAG；中間的NADPH-binding domain 包含活性中心CAVC（C-161和C-164 ）和第180個胺基酸起的GGGDSA序列，除此之外也可發現能和NADP的第二個磷酸根相接觸的鹼性胺基酸R-204 和R-209；C端 FAD binding domain的序列為第 297-298胺基酸序列TS、第302-304胺基酸序列VFA及第306-307胺基酸序列GD，這些位置都跟 FAD binding相關，第321胺基酸序列SSGCMA也屬於FAD binding domain。
A. camphorata TR以pYEX-S1作為表現載體，以S. cerevisiae作為表現宿主。以nickel chelating Sepharose之親合性管柱來純化，可得到37 kDa的A. camphorata TR。從A. camphorata TR跟DTNB的酵素動力學分析，可以得到A. camphorata TR對DTNB的Km值在1740.95 ± 210.57 μM而對於Trx的Km值為2.10 ± 0.45μM。在特性分析上， A. camphorata TR經60 ℃加熱 2 min，還有77 % 的活性，但是隨著加熱時間的增加，酵素活性逐漸喪失，當加熱到 8 min後，則只剩下50 % 左右 的活性。經不同的pH處理，顯示強酸對會讓TR的活性完全喪失。以0.1 imidazole 處理，A. camphorata TR的活性只剩76 %。以chymotrypsin在37 ℃反應10 min後，活性剩下50 %。以trypsin處理5 min後，活性只剩下61 % 左右。

Abstract

The purpose of this thesis is to study antioxidant enzyme. A full length cDNA of 1273 bp encoding a putative thioredoxin reductase (TR) from Antrodia camphorata fruit body was cloned by PCR. Nucleotide sequence analysis of this cDNA revealed that it comprised a complete open reading frame coding for 343 amino acid residues. Analysis of its sequence revealing the distribution by binding domains is as follows：The N-terminal FAD binding domain is a glycine-rich GSGPAG motif at position 29. Then, the central NADPH-binding domain contains an active site comprising the redox-acitive disulfide between C-161 and C-164（motif CAVC）and a glycine –rich GGGDSA motif at position 180. The basic residues R-204 and R-209 that contact the 2′-phosphate group of NADP are also found. The C-terminal FAD binding domain, there are residues 297-298（TS）, 302-304（VFA）, 306-307（GD）, involved in FAD binding, and a glycine-rich SSGCMA motif at position 321.
To further characterize the A. camphorata TR, the coding region was subcloned into an expression vector pYEX-S1 and transformed into S. cerevisiae. The expression of the A. camphorata TR was purified by Ni2+ -nitrilotriacetic acid Sepharose superflow column. The purified enzyme were estimated to be about 37 kDa by 10 % SDS-PAGE. The Km values of A. camphorata TR for DTNB was determined 1740.95 ± 210.57 μM. The Km values of A. camphorata TR for Trx was determined 2.10 ± 0.45 μM. The enzyme retained 77 % activity after heating at 60 ℃ for 2 min. The enzyme activity was inhibited under pH 2.5. The enzyme retained 76 % activity under 0.1 M imidazole treatment. The enzyme showed 50 % activity after 10 min of incubation at 37 ℃ with chymotrypsin. The enzyme showed 61 % activity after 5 min of incubation at 37 ℃ with trypsin.

目錄

中文摘要 ………………………………………………………………...Ⅰ
英文摘要 ………………………………………………………………...Ⅱ
縮寫表 …………………………………………………………………...Ⅳ
壹、 前言………………………………………………………………….1
1.1樟芝……………………………………………………….. …………1
1.2自由基…………………………………………………………….......5
1.3 Thioredoxin system…………………………………… ……………..7
1.4實驗緣起………...……………………………………..……………12
貳、 實驗材料………………………………………………………….. 20
參、 實驗方法……………………………………………………….......26
3.1 樟芝子實體和菌絲體mRNA之萃取…………………...................26
3.2 樟芝子實體和菌絲體cDNA之合成……………………………….27
3.3 A. camphorata TR cDNA之基因選殖……………………...………28
3.4 A. camphorata TR cDNA和表現載體之建構……………….……..32
3.5重組蛋白質的純化……………….……………………………...….35
3.6酵素活性測定……………… …………………………………..…..39
3.7蛋白質之基本特性分析……………….……………………...…….41
肆、 結果與討論………………………………………………………...45
4.1 A. camphorata TR 的基因選殖與序列分………………………….45
4.2 A. camphorata TR的分類 .………………………………...……..47
4.3 A. camphorata TR之純化與表現 ...……………………………..47
4.4 A. camphorata TR酵素動力學分析 ……………..…………...…48
4.5 A. camphorata TR 特性分析 …………………………...………49
參考文獻…………………………………………………………...……..61
附錄……………………………………………………………...………..67

參考文獻

山羽儀兵 釋譯者。三版。植物通論。1976，台北市 正中出版社。
王伯徹、黃仁彰。靈芝與樟芝之研發與是場面面觀。2002，食品工業發展研究所 349 (5)︰3-17。
李學勇。基礎植物學。1990，台北市 國立編譯館。
易希珍。普通植物學。1976，台北市 環球出版社。
Arner, E.S., Holmgren A., 2000. Physiological functions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267, 6102-6109.
Arscott, L.D., Gromer, S., Schirmer, R.H., Becker, K., Williams Jr, C.H., 1997. The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Eescherichia coli. Proc. Natl. Acad. Sci. U S A 94, 3621-3626.
Asahi, T., Bandurski, R.S., Wilson, L.G., 1961. Yeast sulfate-reducing system. II. enzymatic reduction of protein disulfide. J. Biol. Chem. 236, 1830-1835.
Becker, K., Gromer, S., Schirmer, R.H., Muller, S., 2000. Thioredoxin reductase as a pathophysiological factor and drug target. FEBS Journal 267, 6118-6125.

Bjornstedt, M., Kumar, S., Bjorkhem, L., Spyrou, G., Holmgren, A., 1997. Selenium and the thioredoxin and glutaredoxin systems. Biomed. Environ. Sci. 10, 271-279.
Bjornstedt, M., Xue, J., Huang, W., Akesson, B., Holmgren, A., 1994. The thioredoxin and glutaredoxin systems are efficient electron donors to human plasma glutathione peroxidase. J. Biol. Chem. 269, 29382-29384.
Black, S., Harte, E.M., Hudson, B., Wartofsky, L., 1960. A specific enzymatic reduction of l (-) methionine sulfoxide and a related nonspecific reduction of disulfides. J. Biol. Chem. 235, 2910-2916.
Casso, D., Beach, D., 1996. A mutation in a thioredoxin reductase homolog suppresses p53-induced growth inhibition in the fission yeast schizosaccharomyces pombe. Mol. Gen. Genet. 252, 518-529.
Gilberger, T.W., Bergmann, B., Walter, R.D., Muller, S., 1998. The role of the C-terminus for catalysis of the large thioredoxin reductase from Plasmodium falciparum. FEBS Lett. 425, 407-410.
Grant, C.M., 2001. Role of the glutathione/glutaredoxin and thioredoxin systems in yeast growth and response to stress conditions. Mol. Microbiol. 39, 533-541.
Holmgren, A., 1979. Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide. J. Biol. Chem. 254, 9627-9632.

Holmgren, A., Bjornstedt, M., 1995. Thioredoxin and thioredoxin reductase. Methods Enzymol. 252, 199-208.
Jeon, S.J., Ishikawa, K., 2002. Identification and characterization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1. Eur. J. Biochem. 269, 5423-5430.
Kehrer, J.P., 1993. Free radicals as mediators of tissue injury and disease. Crit. Rev. Toxicol. 23, 21-48.
Muro, Y., Ogawa, Y., Kato, Y., Hagiwara, M., 1998. Autoantibody to thioredoxin reductase in an ovarian cancer patient. Biochem. Biophys. Res. Commun. 242, 267-271.
Lee, D.Y., Ahn, B.Y., Kim, K.S., 2000. A thioredoxin from the hyperthermophilic archaeon methanococcus jannaschii has a glutaredoxin-like fold but thioredoxin-like activities. Biochemistry 39, 6652-6659.
Lennon ,B.W., Williams Jr, C.H., 1997. Reductive half-reaction of thioredoxin reductase from Eescherichia coli. Biochemistry 36, 9464-9477.
Lennon, B.W., Williams Jr C.H., 1995. Effect of pyridine nucleotide on the oxidative half-reaction of Eescherichia coli thioredoxin reductase. Biochemistry 34, 3670-3677.


Matthews, J.R., Wakasugi, N., Virelizier, J.L., Yodoi, J., Hay, R.T., 1992. Thioredoxin regulates the DNA binding activity of NF-kappa B by reduction of a disulphide bond involving cysteine 62. Nucleic Acids Res. 20, 3821-3830.
Miranda Vizuete, A., Damdimopoulos, A.E., Pedrajas, J.R., Gustafsson, J.A., Spyrou, G., 1999. Human mitochondrial thioredoxin reductase cDNA cloning, expression and genomic organization. Eur. J. Biochem. 261, 405-412.
Moore, E.C., Reichard, P., Thelander, L., 1964a. Enzymatic synthesis of deoxyribonucleotides.V. purification and properties of thioredoxin reductase from Eescherichia coli B. J. Biol. Chem. 239, 3445-3452.
Moore, E.C., Reichard, P., Thelander, L., 1964b. Enzymatic synthesis of deoxyribonucleotides.V. purification and properties of thioredoxin reductase from Eescherichia coli B. J. Biol. Chem. 239, 3445-3452.
Nakamura, H., Nakamura, K., Yodoi, J., 1997. Redox regulation of cellular activation. Annu. Rev. Immunol. 15, 351-369.
Nordberg, J., Arner, E.S., 2001. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med. 31, 1287-1312.
Pedrajas, J.R., Kosmidou, E., Miranda Vizuete, A., Gustafsson, J.A., Wright, A.P.H., Spyrou, G., 1999. Identification and functional characterization of a novel mitochondrial thioredoxin system in saccharomyces cerevisiae. J. Biol. Chem. 274, 6366-6373.
Pigiet, V.P., Conley, R.R., 1977. Purification of thioredoxin, thioredoxin reductase, and glutathione reductase by affinity chromatography. J. Biol. Chem. 252, 6367-6372.
Reid, T.M., Loeb, L.A., 1993. Effect of DNA-repair enzymes on mutagenesis by oxygen free radicals. Mutat. Res. 289, 181-186.
Russel, M., Model, P., 1988. Sequence of thioredoxin reductase from Eescherichia coli. relationship to other flavoprotein disulfide oxidoreductases. J. Biol. Chem. 263, 9015-9019.
Stefankova, P., Perecko, D., Barak, I., Kollarova, M., 2006. The thioredoxin system from streptomyces coelicolor. J. Basic. Microbiol. 46, 47-55.
Sun, Q.A., Wu, Y., Zappacosta, F., Jeang, K.T., Lee, B.J., Hatfield, D.L., Gladyshev, V.N., 1999. Redox regulation of cell signaling by selenocysteine in mammalian thioredoxin reductases. J. Biol. Chem. 274, 24522-24530.
Tamura, T., Stadtman, T.C., 1996 A new selenoprotein from human lung adenocarcinoma cells: Purification, properties, and thioredoxin reductase activity. Proc. Natl. Acad. Sci. U S A 93, 1006-1011.
Tarrio, N., Diaz Prado, S., Cerdan, M.E., Gonzalez Siso, M.I., 2004. Isolation and characterization of two nuclear genes encoding glutathione and thioredoxin reductases from the yeast kluyveromyces lactis. Biochim. Biophys. Acta. 1678, 170-175.

Tsang, M.L., Schiff, J.A., 1976. Sulfate-reducing pathway in Eescherichia coli involving bound intermediates. J. Bacteriol. 125, 923-933.
Wang, P.F., Veine, D.M., Ahn, S.H., Williams Jr, C.H., 1996. A stable mixed disulfide between thioredoxin reductase and its substrate, thioredoxin: Preparation and characterization. Biochemistry 35, 4812-4819.
Williams Jr, C.H., Arscott, L.D., Muller, S., Lennon, B.W., Ludwig, M.L., Wang, P.F., et al., 2000. Thioredoxin reductase two modes of catalysis have evolved. Eur. J. Biochem. 267, 6110-6117.
Williams Jr, C.H., 1995. Mechanism and structure of thioredoxin reductase from Eescherichia coli. FASEB J. 9, 1267-1276.
Wood, Z.A., Schroder, E., Robin Harris, J., Poole, L.B., 2003. Structure, mechanism and regulation of peroxiredoxins. Trends Biochem. Sci. 28, 32-40.