臺灣博碩士論文加值系統

當重金屬鎘進入細胞後，會造成大部分的胞內酵素失活，使得細胞的正常代謝反應受到干擾，導致細胞生長受阻。然而植物或裂殖性酵母菌中的植物螯合素合成 (phytochelatin synthase, PCS) 反而會受到鎘的活化，以glutathione (GSH) 為基質合成植物螯合素 (phytochelatin, PC)。PC可結合進入細胞內的鎘，先短暫形成分子量較小的Cd-PC複合體 (LMW)，然後由液泡膜上HMT1 (heavy metal tolerance gene translated protein) 運送入液泡內，並加上硫離子形成較大的Cd-PC S2- 複合體 (HMW)。藉以上過程可將進入細胞內的重金屬鎘安全地隔離。
本論文以PCS為主要研究對象，首先改進PCS的活性分析，發現加入磷酸可以增強PCS的活性達數十倍；並以此分析法，重新改進PCS的純化步驟。 然後進一步製備單株抗體工具，加上各種方法，確定純化所得蛋白質確實為PCS，所用的方法分別為： (1) 純化流程中各步驟所得之PCS，以抗PCS的單株抗體 (Anti-PCS) 進行免疫呈色均可得正反應；(2) 所純化PCS原態分子量為46 kD，與由基因選殖推算者相符 (Clements et al, 1999)； (3) PCS單株抗體可抑制PCS活性。 其次是釐清PCS的催化機制，發現：(1) 磷酸與Cd都可促進PCS的活性；(2) PCS以GSH當作基質，而不能以 gEC當作基質；(3) NaCl會抑制PCS活性。 其中磷酸能促進PCS活性的發現，迄今無人報告，而且效果非常大，所以本論文試圖以多種方式釐清磷酸對於PCS的影響。

When fission yeast was exposed to heavy metals, most enzymes in the cell were inactivated, and the metabolism of the cell retarded. However, the activity of phytochelatin synthase (PCS) in fission yeast cell was activated, and catalyzed the biosynthesis of phytochelatins (PC) using glutathione (GSH) as the substrate. PC molecules chelated the invading cadmium ions, produced a lower molecular weight Cd-PC complex (LMW), which was then transported by a transporter, HMT1, into the vacuole. The LMW complex was transformed into the higher molecular weight Cd-PC-S2+ complex (HMW) and was sequestered in the vacuole.
In this study, we improved the assay system of PCS by incorporating phosphate in the substrate mixture. The activity of PCS increased at least ten folds when measuring the phytochelatins produced. Using this assay method, we had refined the purification procedure for PCS. The purified enzyme was then used to prepare monoclonal antibodies against PCS (Anti-PCS). On Western blot, the antibody bound a clear band having the molecular mass of 46 kD, which is consistent with the molecular mass deduced from the cDNA of PCS (Clements et al, 1999). The enzyme activity was inhibited by Anti-PCS. When examined the catalytic behavior of PCS, we found that both phosphate and Cd increased the activity. On the other hand, gEC was not a good substrate, and NaCl inhibit PCS activity.

謝誌………………………………………………………………………...v
中文摘要…………………………………………………………………..vi
英文摘要………………………………………………………………….vii
縮寫表……………………………………………………………………viii
第一章 緒論……………………………………………………………….1
1.1 重金屬……………………………………………………………..1
1.2 重金屬對生物體所造成的傷害…………………………………..3
1.2.1 重金屬離子對細胞的損害…………………………………………..….3
1.2.2 重金屬離子對人類造成的傷害…………………………………..…….3
1.3 生物體中的重金屬隔離機制……………………………………..4
1.3.1 金屬硫蛋白……………………………………….…………….4
1.3.2 植物螯合素…………………………………………………..………….5
1.3.3 植物螯合素合成…………………………………………………..….6
1.3.4 植物螯合素合成之基因序列………………………………………...7
1.3.5植物螯合素合成可能之催化機制………………………………7
1.4 重金屬污染解決之道與實驗緣起………………………………..9
圖1-1 至圖1-6……………………………………………………....12
第二章 材料與方法……………………………………………………..18
2.1 酵母菌培養………………………………………………………18
2.1.1 菌種………………………………………………………………..…...18
2.1.2 洋菜膠培養法………………………………………………………….18
2.1.3 液態培養法……………………………………………….…..19
2.2 酵母菌的粗抽取液………………………………………………21
2.2.1 玻璃砂小量萃取法…………………………………..………...21
2.2.2 玻璃砂大量萃取法………………………………………….....22
2.3 生物化學分析法…………………………………………………23
2.3.1 蛋白質定量法……………………………………………………….....23
2.3.2 蛋白質 N-端序列決定法 (一)…………………………………...…...24
2.3.3 蛋白質 N-端序列決定法 (二)……………………………..……25
2.4 一般化學檢定法…………………………………………………27
2.4.1 鎘離子的測定……………………………………………….………....27
2.4.2 硫醇基的測定………………………………………….……...28
2.5 管柱層析法……………………………..………………………..29
2.5.1 膠體過濾法…………………………………………….……………....29
2.5.2 離子交換法………………………………………………………...…..31
2.5.3 金屬螯合層析法…………………………………………………...…..32
2.5.4 Hydroxylapatite 層析法……………………….…………………..….32
2.5.5 親和性層析法 (Protein A-Sepharose CL-4B)..…….……………...33
2.5.6 高效液相層析法 (HPLC) 基本操作…………...…….…………35
2.6 電泳檢定法………………………………………………………37
2.6.1 不連續膠體電泳 (native-PAGE)………………………………….…37
2.6.2 SDS 膠體電泳…………………………………………………….…..40
2.6.3 膠體染色法…………………………………………………………....41
2.6.4 膠片乾燥法………………………………………………..…...44
2.7植物螯合素合成純化法……………………………………….45
2.7.1 PCS活性分析………………………………………..………..45
2.8 專一性多源抗體製備…...……………………………………….47
2.8.1抗原之製備…...…………………………………………………..…….47
2.8.2動物免疫…………...……………………………………………..…….48
2.8.3免疫球蛋白之純化…...……………………………………..….49
2.9 單株抗體之製備…...…………………………………………….51
2.9.1小白鼠免疫…...……………………………………………………..….51
2.9.2 細胞融合……..……………………………………………………..….51
2.9.3 細胞保存法…...…………………………………………………….….55
2.9.4 單株抗體的生產…...……………………………………………57
2.10 酵素免疫分析法…...……………………………………….….58
2.11酵素免疫染色法.…...……………………………………….….60
第三章 結果與討論……………………………………………………..63
3.1 植物螯合素合成的純化………………………………………63
3.1.1植物螯合素合成的活性測定法………………………………..……63
3.1.2 植物螯合素合成專一性抗體製備……………………………….....64
3.1.3 植物螯合素合成之純化…………..……………………………..….65
3.1.4 PCS 的性質檢定……………………………………………....68
3.2 植物螯合素合成催化機制的探討……………………………70
3.2.1 鎘離子可活化 PCS 活性……………………………………….…...70
3.2.2 鎘離子濃度對 PCS 活性的影響…………………………….……...70
3.2.3 磷酸可活化 PCS 活性………………………………………..……..70
3.2.4 磷酸、鎘離子與 GSH 加入次序會影響PCS 活性……………….70
3.2.5 Cd-GS2 對 PCS 活性的影響……………………….…………….....71
3.2.6 gEC 對 PCS 活性的影響……………………….…………………...71
3.2.7 NaCl 對 PCS 活性的影響……………………….….………...72
圖3-1 至圖3-23……………………………………………………..….73
表 3-1…………………………………………………………………….83
第四章 結論……………………………………………………………...97
參考文獻…..……………………………………………………………...99
問答錄……..…………………

吳建興 (2000) 裂殖性酵母菌中植物螯合素合成的分離與性質研究。博士論文。國立台灣大學農業化學系，台北。
吳其真 (1998) 甘藷澱粉磷解之生化及免疫學研究。碩士論文。國立台灣大學農業化學系，台北。
莊榮輝 (1988) 水稻蔗糖合成之研究。博士論文。國立台灣大學農業化學系，台北。
Bernhard WR, Kägi JH (1987) Purification and characterization of a typical cadmium-binding polypeptides from Zea mays. Experientia Suppl 52: 309-315
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254
Brooks RR (1998) Plants that hyperaccumulate heavy metals. CAB International, UK
Charles C.Allain, Lucy S. Poon, Cicely S. G. Chan, W. Richmond, and Paul C. Fu (1974) Enzymatic determination of total serum cholesterol. Clin Chem 20/4: 470-476
Chen J, Zhou J, Goldsbrough PB (1997) Characterization of phytochelatin synthase from tomato. Physiol Plant 101: 165-172
Clemens S, Kim EJ, Neumann D, Schroeder JI (1999) Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast. EMBO J 18: 3325-3333
Cobbett CS (1999) A family of phytochelatin synthase genes from plant, fungal and animal species. Trends Plant Sci 4: 335-337
Collins YE, Stotzky G (1989) Factors affecting the toxicity of heavy metals to microbes. In Beveridge TJ, Doyle RJ, eds, Metal ions and bacteria, John Wiley and Sons, New York, pp 32-35
Deutscher MP (1990) Rethinking your purification procedure. Methods Enzymol 182: 779-780
Dixon M, Webb EC, Thorne CJR, Tipton KF (1979) Enzymes 3rd ed. Academic Press Inc., New York, pp 126-126
Donlon J.and P.F.Fottrell (1971) Quantitative determination of intestinal peptide hydrolase activity using L-amino acid oxidase. Clinic Chimica Acta 33: 345-350
Fuente JM, Ramirez-Rodriguez V, Cabrera-Ponce JL, Herrera-Estrella L (1997) Aluminum tolerance in transgenic plants by alternation of citrate synthesis. Science 276: 1566-1568
Furey WF, Robbins AH, Clancy LL, Winge DR, Wang BC, Stout CD (1986) Crystal structure of Cd, Zn metallothionein. Science 231: 704-710
Grill E (1987) Phytochelatins, the heavy metal binding peptides of plants: characterization and sequence determination. Experientia Suppl 52: 317-322
Grill E, Gekeler W, Winnacker EL, Zenk MH (1986) Homo-phytochelatins are metal-binding peptides of homo-glutathione containing Fabales. FEBS Lett 205: 47-50
Grill E, Loffler S, Winnaccker E, Zenk MH (1989) Phytochelatins, the heavy metal-binding peptides of plants, are synthesized from glutathione by a specific g-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proc Natl Acad Sci USA 86: 6838-6842
Grill E, Winnacker EL, Zenk MH (1985) Phytochelatins: the principle heavy-metal complexing peptides of higher plants. Science 230: 674-676
Grill E, Winnacker EL, Zenk MH (1991) Phytochelatins. Methods Enzymol 205: 333-341
Ha SB, Smith AP, Howden R, Dietrich WM, Bugg S, O''Connell MJ, Goldsbrough PB, Cobbett CS (1999) Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe. Plant Cell 11: 1153-1164
Hayashi Y, Nakagawa CW, Murasugi A (1986) Unique properties of Cd-binding peptides induced in fission yeast, Schizosaccharomyces pombe. Environ Health Perspect 65: 13-19
Hayashi Y, Nakagawa CW, Mutoh N, Isobe M, Goto T (1991) Two pathways in the biosynthesis of cadystins (g-EC)nG in the cell- free system of the fission yeast. Biochem Cell Biol 69: 115-121
Howden R, Goldsbrough PB, Andersen CR, Cobbett CS (1995) Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol 107: 1059-1066
Howden R, Andersen CR, Goldsbrough PB, Cobbett CS (1995b) A cadmium-sensitive, glutathione-deficient mutant of Arabidopsis thaliana. Plant Physiol 107: 1067-1073
Jazwinski SM (1990) Preparation of extracts from yeast. Methods Enzymol 182: 154-174
Jones MM and Vaughn WK (1978) HSAB theory and acute metal ion toxicity and detoxification processes. J Inorg Nucl Chem 40:2081
Karin M (1985) Metallothioneins: proteins in search of function. Cell 41: 9-10
Klapheck S, Chrost B, Starke J, Zimmermann H (1992) g-Glutamylcysteinylserine: a new homolog of glutathione in plants of the family Poaceae. Bot Acta 105: 174-179
Kondo N, Imai K, Isobe M, Goto T, Murasugi A, Wada-Nakagawa C, Hayashi, Y (1984) Tetrahedron Lett. 25, 3869-3872
Kramer U, Cotter-Howells JD, Charnock JM, Banker AJM, Smith JAC (1996) Free histidine as a metal chelator in plants that accumulate nickel. Nature 379: 635-638
Kubota H, Sato K, Yamada T, Maitani T (2000) Phytochelatin homologs induced in hairy roots of horseradish. Phytochemistry 53: 239-245
Liang ZY, Pilon-Smits EA, Jouanin L, Terry N (1999) Overexpression of glutathione synthetase in indian mustard enhances cadmium accumulation and tolerance. Plant Physiol 119: 73-80
Lohr D (1988) Isolation of yeast nuclei and chromatin for studies of transcription related processes. In Campbell I, Duffus JH, eds, Yeast - a practical approach, IRL Press Limitted, Oxford, pp 125-145
Ma JF, Hiradate S, Matsumoto H (1997) Detoxifying aluminum with buckwheat. Nature 390: 569-570
Ma JF, Hiradate S, Matsumoto H (1998) High aluminium resistance in buckwheat: II. Oxalic acid detoxifies aluminium internally. Plant Physiol (Rockville) 117: 753-759
Ma Z, Miyasaka SC (1998) Oxalate exudation by taro in response to Al. Plant Physiol 118: 861-865
Maret W (1994) Oxidative metal release from metallothionein via zinc-thiol/disulfide interchange. Proc Natl Acad Sci USA 91: 237-241
Margoshes M, Vallee BL (1959) A cadmium protein from equine kidney cortex. J Am Chem Soc 79: 4813-4814
Meuwly P, Thibault P, Rauser WE (1993) g-Glutamylcysteinylglutamic acid - a new homologue of glutathione in maize seedlings exposed to cadmium. FEBS Lett 336: 472-476
Mitts MR, Grant DB, Heideman W (1990) Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein. Mol Cell Biol 10: 3873-3883
Moore S, Stein WH (1963) Chromatographic determination of amino acid by the use of automatic recording equipment. Methods Enzymol 6: 819-931
Nieboer E, Richardson DHS (1980) The replacement of the nondescript term heavy metals by a biological and chemically significant classification of metal ions. Environ Pollut Ser. B: 1-3
Olena KV, Stephane M, Lu YP (2000) Mechanism of heavy metal ion activation of phytochelatin (PC) synthase. J Biol Chem 275: 31451-31459
Ortiz DF, Kreppel L, Speiser DM, Scheel G, McDonald G, Ow DW (1992) Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter. EMBO J 11: 3491-3499
Ortiz DF, Ruscitti T, McCue KF, Ow DW (1995) Transport of metal-binding peptides by HMT1, a fission yeast ABC-type vacuolar membrane protein. J Biol Chem 270: 4721-4728
Pearson RG (1968) Hard and soft acids and bases, HSAB, Part1, J Chem Educ 45: 643
Rabinowitz JC (1978) Analysis of acid-labile sulfide and sulfhydryl groups. Methods Enzymol 53: 273-275
Reese RN, White CA, Wing DR (1999) Cadmium-sulfide crystallites in Cd-(gammaEC)nG peptide complexes from tomato. Plant Physiol 98: 225-229
Rennenberg H (1989) Aspect of glutathione function and metabolism in plants. Plant Mol Biol 140: 279-292
Roberts CJ, Raymond CK, Yamashiro CT, Stevens TH (1991) Methods for studying the yeast vacuole. Methods Enzymol 194: 644-661
Rose AH, Veazey FJ (1988) Yeast a practcal approach - Separation of organelles from lysates. IRL, Oxford
Salt DE, Smith RD, Raskin I (1998) Phytoremediation. Annu Rev Plant Physiol Plant Mol Biol 49: 643-668
Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368-379
Schwartz JH, Greenberg SM (1987) Molecular mechanisms for memory: second-messenger induced modifications of protein kinases in nerve cells. Annu Rev Neurosci 10: 459-476
Speiser DM, Ortiz DF, Kreppel L, Scheel G, McDonald G, Ow DW (1992) Purine biosynthetic genes are required for cadmium tolerance in Schizosaccharomyces pombe. Mol Cell Biol 12: 5301-5310
Steffens JC (1990) The heavy metal-binding peptides of plants. Plant Mol Biol 41: 553-575
Vatamaniuk OK, Mari S, Lu YP, Rea PA (1999) AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution. Proc Natl Acad Sci USA 96: 7110-7115
Wu JS, Sung HY, Juang RH (1995) Transformation of cadmium-binding complexes during cadmium sequestration in fission yeast. Biochem Mol Biol Int 36: 1169-1175
Zenk MH (1996) Heavy metal detoxification in higher plants - a review. Gene 179: 21-30