臺灣博碩士論文加值系統

植物的L型澱粉磷解酶 (L-form starch phosphorylase, L-SP) 在其分子中央，較動物的肝醣磷解脢多出一段由78個胺基酸組成的片段 (L78)。因為組成及結構特殊的關係，L78極易斷裂，造成L-SP分子裂解成兩群分子量相近的片段 (約50 kDa, F50s)。已知甘藷塊根加熱至45oC會使L-SP產生規律之階段性降解，隨著加熱反應時間的增加，抗體Anti-L78無法辨識任何的L-SP，加熱亦造成甘藷切片之糊化現象。此種L-SP降解似乎具有調節性。進一步觀察發現，在加熱糊化初期，糊化區中幾乎不存在完整的L-SP 110 kDa分子，大部分都降解為特定的F50片段。由於產生特定F50片段的現象只在加熱後第16小時觀察得到，第24小時後L-SP會快速降解，因此我們推測加熱產生的糊化初期 (加熱16小時)，會引發專一性L78降解機制；隨著加熱時間延長至24小時後，糊化情形加劇導致胞器破裂，釋放更多蛋白酶，對L-SP產生隨機且快速的降解。本研究取16小時之糊化樣品，經硫酸銨分劃後以膠體過濾法分離，再經蛋白酶活性分析，得知此專一性降解蛋白酶位於20-40% 硫酸銨分劃。目前已知硫酸銨分劃中主要有四個以gelatin為基質的蛋白酶活性，

The L-form starch phosphorylase (L-SP) is abundant in sweet potato (Ipomoea batatas) roots with a higher molecular mass of 110 kDa. In the middle of sweet potato L-SP, a 78-amino acid insert (L78) was found when compared with its counter part in animal cells, glycogen phosphorylase. L78 was susceptible to multiple proteolytic modification because of its unique amino acid sequence and molecular structure. L-SP was cleaved into two groups of fragments having the molecular mass around 50 kDa (F50s) by these modifications. If the slices of sweet potato roots were heated at 45oC, we could observe the change of the mobility of L-SP on native gel electrophoresis. During the heating process, the sweet potato splices were subjected to gelatinization and the controlled proteolytic modification began. At the beginning of gelatinization (16 h heating), the 110 kDa L-SP almost disappeared on the native or SDS gel. Gelatinization might therefore trigger amechanism to cleave L78 specifically. After longer period of incubation (24 h), L-SP was degraded rapidly in an uncontrolled process. A L-SP specific protease might be induced by the controlled heating process which led to the gelatinization of the sweet potato tissue. Using ammonium sulfate fractionation and gel filtration, the protease activities were isolated and partially purified.

摘要 1
Abstract 2
一、前言 3
1.1 澱粉磷解酶 3
1.2 植物逆境 (plant stress) 6
1.3 澱粉粒 (Starch granule) 12
1.4 植物中的蛋白質降解 15
1.5 研究動機 24
二、材料與方法 25
2.1電泳檢定法 25
2.1.1 原態膠體電泳 25
2.1.2 SDS膠體電泳 28
2.1.3 可溶性澱粉親和性電泳 30
2.1.4 製備式電泳與電泳溶離 31
2.1.5 甘藷塊根澱粉磷解酶合成澱粉活性染色法 33
2.1.6 Zymography 34
2.1.7 膠體染色法 38
2.1.8 膠片乾燥法及護貝 40
2.1.9 蛋白質電泳轉印法 41
2.2一般分析法 42
2.2.1蛋白質定量 42
2.2.2甘藷塊根澱粉磷解酶合成澱粉活性分析(添加醣引子) 43
2.2.3酵素免疫染色法 45
2.3 管柱色層分析法 (Chromatography) 47
2.3.1管柱色層層析法之基本操作 47
2.3.2膠體過濾法 49
2.3.3離子交換法 50
2.4 甘藷塊根L型澱粉磷解酶製備法 51
2.4.1酵素粗抽取及硫酸銨分劃 51
2.4.2甘藷塊根L型澱粉磷解酶純化法 53
2.5甘藷45oC溫度處理實驗 54
2.6甘藷塊根20S proteasome純化 55
三、結果與討論 59
3.1甘藷澱粉磷解酶之階段性降解 59
3.2 45oC處理之甘藷切片粗抽液對L-SP之影響 59
3.2.1內生性L-SP降解情形 59
3.2.2外加L-SP之降解情形 61
3.2.3可溶性澱粉親和性分析 63
3.3 外加proteasome對於純化之L-SP影響 63
3.4 加熱甘藷切片之蛋白酶純化 64
3.4.1甘藷切片45oC加熱不同時間之蛋白酶活性分析 64
3.4.2甘藷切片45oC加熱16 h之蛋白酶活性分析 64
3.4.3 16 h蛋白酶純化與分析 65
四、總結 95
五、參考文獻 97
答問錄 103

陳翰民 (1997) 甘藷澱粉磷解酶構造與功能之研究 博士論文 國立臺灣大學 臺北
蔡豐仁 (1998) 降解甘薯塊根澱粉磷解梅之蛋白酶：其純化、性質鑑定與免疫組織定位 博士論文 國立臺灣大學 臺北
吳其真 (1998) 甘藷澱粉磷解酶之生化及免疫學研究 碩士論文 國立臺灣大學 臺北
張世宗 (1999) 甘藷塊根Chaperonin及Proteasome之分離與性質研究 博士論文 國立臺灣大學 臺北
陳安娜 (2001) 甘藷塊根澱粉磷解酶降解路徑的探討與Proteasome的結合關係 碩士論文 國立臺灣大學 臺北
林怡岑 (2003) 甘藷塊根澱粉磷解酶與Proteasome之結合與降解關係 碩士論文 國立臺灣大學 臺北
周宜旻 (2005) 甘藷塊根澱粉磷解酶之蛋白質交互作用 碩士論文 國立臺灣大學 臺北
葉昭圻 (2005) 甘藷塊根澱粉磷解脢高溫下階段式降解之探討 碩士論文 國立臺灣大學 臺北
楊光華 (2005) 甘藷塊根L型澱粉磷解酶激酶之純化與性質分析 博士論文 國立臺灣大學 臺北
A.Buleón, P. Colonna, V. Planchot, S. Ball (1998) Starch granules: structure and biosynthesis. International Journal of Biological Macromolecules 23: 85–112
Alan M. Myers, Matthew K. Morell, Martha G. James, and Steven G. Ball (2000) Recent Progress toward Understanding Biosynthesis of the Amylopectin Crystal. Plant Physiology 122: 989–997
Alfonso, M. et al. (2001) Unusual tolerance to high temperatures in a new herbicide-resistant D1 mutant from Glycine max (L.) Merr. Cell cultures deficient in fatty acid desaturation. Planta 212: 573-582
Andreas S (2004) A cut above the rest: the regulatory function of plant proteases. Planta 220: 183-197
Barrett A J (1986) The classes of proteolytic enzymes. In: Plant Proteolytic Enzymes, M J Dalling, ed (Boca Raton, FL: CRC Press), pp 1-16
Barrett A J (1994) Classification of peptidases. Methods Enzymol. 244: 1-15
Beynon RJ, Bond JS (1989) Proteolytic enzymes. In: Plant Proteolytic Enzymes, M J Dalling, ed (Boca Raton, FL: CRC Press), pp 1-16
Brisson N, Giroux H, Zollinger M, Camirand A, Simard C (1989) Maturation and subcellular compartmentation of potato starch phosphorylase. Plant Cell 1: 559-566
Bushnell T, Bushnell D, Jagendorf A T (1993) A purified zinc protease of pea chloroplasts, EP1, degrades the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Plant Physiol 103: 585-591
Callis J (1995) Regulation of Protein Degradation. The Plant Cell 7: 845-857
Chatakanoda P, Chinachoti P, Sriroth K, Piyachomkwan K, Chotineeranat S, Tang HR, Hills B (2003) The influence of time and conditions of harvest on the functional behavior of cassava starch-a proton NMR relaxation study. Carbohydrate Polymers 53: 233-240
Ciechanover A (1994) The ubiquitin-proteasome proteolytic pathway. Cell 79: 13-21
Correns C (1901). Bibl. Bot 53: 1-161
Coux O, Tanaka K, Goldberg AL (1996) Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem 65: 801-847
Crawford NM (1995) Nitrate: Nutrient and signal for plant growth. Plant Cell 7: 859-868
Dat JF, Foyer CH, Scott IM (1998) Changes in salicylic acid and antioxidants during induction of thermotolerance in mustard seedlings. Plant Physiol 118: 1455-1461
de Barros E, Larkins BA (1990) Purification and characterization of zein-degrading protease from endosperm of germinating maize seeds. Plant Physiol 94: 297-303
Dong Yul Sung, Fatma Kaplan, Kil-Jae Lee and Charles L. Guy (2003) Acquired tolerance to temperature extremes. Trends in Plant Science 8 (4): 179-187
Donovan J (1979) Phase transitions of the starch-water system. Biopolymers 18: 263-275
Egas C, Lavoura N, Resende R, Brito RMM, Pires C, Pedroso de Lima MC, Faro C (2000) The saposin-like domain of the plant aspartic proteinase precursor is a potent inducer of vesicle leakage. J Biol Chem 275: 38190-38196
Fincher G (1989) Molecular and cellular biology associated with endosperm mobilization in germinating cereal grains. Annu Rev. Plant Physiol. Plant Mol. Biol 40: 305-346
Frank F (1986) Unfrozen water : Yes ; unfreezable water : hardly ; bound water : certainly not. Cryo-Letters 7: 207
Fukui T (1983) Plant phosphorylase: structure and function. In Akazawa T, Asahi T and Imaseki H. eds. The New Frontiers in Plant Biochemistry, Japan Scientific Societies Press, Tokyo, pp. 71-82
Goldberg AL, Rock KL (1992) Proteolysis, proteasomes and antigen presentation. Nature 357: 375-379
Gong M, Li X-J, Dai X, Tian M, Li Z-G (1997) Involvement of calcium and calmodulin in the acquisition of HS induced thermotolerance in maize seedlings. J Plant Physiol 150: 615-621
Goodfellow VJ, Solomonson LP, Oaks A (1993) Characterization of a maize root proteinase. Plant Physiol 101: 415-419
Han-Min Chen, Shih-Chung Chang, Chi-Chen Wu, Ting-Shen Cuo, Jiann-Shing Wu and Rong-Huay Juang (2002) Regulation of the catalytic behaviour of L-form starch phosphorylase from sweet potato roots by proteolysis. Physiologia Plantarum 114 (4): 506-515.
Hensel LL, Ganoth V, Baumgerten DA, Bleecker AB (1993) Development and age-regulated processes that influence the longevity and senescence of photosynthetic tissues in Arabidopsis. Plant Cell 5: 553-564
Hilt W, Wolf DH (1996) Proteasomes: destruction as a programme. Trands Biochem Sci 21: 96-102
Hough R, Pratt G, Rechsteiner M (1987) Purification of two high molecular weight proteases from rabbit reticulocyte lysate. J Biol Chem 262: 8303-8313
Huber JL, Huber SC, McMichael RW, Redinbaugh MG, Campbell WH (1993) Modulation of nitrate reductase activity by protein phosphorylation. Curr. Top. Plant Biochem. Physiol 12: 7-8
Hugly, S. et al. (1989) Enhanced thermal tolerance of photosynthesis and altered chloroplast ultrastructure in a mutant of Arabidopsis deficient in lipid desaturation. Plant Physiol. 90: 1134-1142
Jonak, C. et al. (1996) Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought. Proc. Natl. Acad. Sci. USA 93: 11274-11279
Jordá L, Coego A, Conejero V, Vera P (1999) A genomic cluster containing four differentially regulated subtilisin-like processing protease genes in tomato plants. J Biol Chem 274: 2360-2365
Kaiser WM, Huber SC (1994) Posttraslational regulation of nitrate reductase in higher plants. Plant Physiol 106: 817-821
Koehler SM, Ho TD. (1990a) A major gibberellic acid-induced barley aleurone cysteine proteinase which digests hordein. Plant Physiol. 94: 251-258
Koehler SM, Ho TD. (1990b) Hormonal regulation, processing and secretion of cysteine proteinases in barley aleurone layers. Plant Cell. 2:769-783
Levitt, J (1980). Responses of Plants to Environmental Stresses, Vol. 1: Academic Press. New York.
Li S, Dickinson LC, Chinachoti P (1998) Mobility of ‘unfreezable’ and ‘freezable’ water in waxy corn starch by 2H and 1H HMR. Journal of Agricultural and Food Chemistry 46: 62-71
Li X –Z, Oaks A (1993) Induction and turnover of nitrate reductase in Zea mays. Influence of NO3–. Plant Physiol 102: 1251-1257
Lichtenthaler, HK. (1988) In vivo chlorophyll fluorescence as a tool for stress detectionin plants. In Applications of Chlorophyll Fluorescence., Ed: 129-142.
Lin YH, Chu HH (1989) Endopeptidases of sprouts and resting roots of sweet potato (Ipomoea batatas [L.] Lam. cv. Tainong 57). J Chinese Biochem Soc 18. 18-28
Lohman KN, Gan S, John MC, Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiol Plant 92: 322-328
Małgorzata Grudkowska and Barbara Zagdańska (2004) Multifunctional role of plant cysteine proteinases. Acta Biochimica Polonica 51(3): 609-624
Maurizi M R, Clark W P, Kim S H, Gottesman S (1990) ClpP represents a unique family of serine proteases. J Biol Chem 265: 12456-12552
Mitsuhashi W, Oaks A (1994) Deveolpment of endopeptidase activities in maize (Zea mays L.) endosperms. Plant Physiol 104: 401-407
Mori H, Tanizawa K, Fukui T (1991) Potato tuber Type H phosphorylase isozyme: molecular cloning, nucleotide sequence and expressison of a full-length cDNA in Escherichiac coli. J Biol Chem 266: 18446-18453
Mori H, Tanizawa K, Fukui T (1993) A chimeric a-glucan phosphorylase of plant type L and H isozymes. J Biol Chem 268: 5574-5581
Nakano K, Fukui T (1986) The Complete Amino Acid Sequence of Potato a-Glucan Phosphorylase. Journal of Biological Chemistry.261: 8230-8236
Orlowski M (1990) The multicatalytic proteinase complex, a major extralysosomal proteolytic system. Biochemistry 29: 10289-10297
Pan D, Nelson O.E. (1984). A Debranching Enzyme Deficiency in Endosperms of the Sugary-1 Mutants of Maize. Plant Physiol 74: 324-328
Payie KG, Tanaka T, Gal S, Yada RY (2003) Construction, expression and characterization of a chimaeric mammalian-plant aspartic proteinase. Biochem J 372: 671-678
Qi X, Chen R, Willson KA, Tan-Willson AL (1994) Characteriztion of a soybean b-conglycinin-degrading protease cleavage site. Plant Physiol 104: 127-133
Ramalho-Santos M, Veríssimo P, Cortes L, Samyn B, Van Beeumen J, Pires E, Faro C (1998) Identification and proteolytic processing of procardosin A. Eur J Biochem 225: 133-138
Rivett A J (1993) Proteasomes: Multicatalytic proteinase complexes. Biochem J 291: 1-10
Rojas A, Almoguera C, Jordano J (1999) Transcriptional activation of a heat shock gene promoter in sunflower embryos: synergism between ABI3 and heat shock factors Plant J 20: 601-610
Ryan C, walker-Simmons M (1981) Plant proteinases. In: The Biochemistry of Plants, Marcus A (ed), New York: Academic Press, pp. 321-349
Sangwan, V. et al. (2002) Opposite changes in membrane fluidity mimic cold and heat stress activation of distinct plant MAP kinase pathways. Plant J. 31: 629-638
Schaffer MA, Fischer RL. (1990) Transcriptional activation by heat and cold of a thiol protease gene in tomato. Plant Physiol. 93: 1486-1491
Schett G, Steiner CW, Groger M, Winkler S, Graninger W, Smolen J, Xu Q, Steiner G (1999) Activation of Fas inhibits heat induced activation of HSF1 and upregulation of HSP70. FASEB J 13: 833-842
Schmidt G, Mishkind M (1983) Rapid degradation of unassembled rebulose-1,5-bisphosphate carboxylase small subunits in chloroplasts. Proc Natl Acad Sci. USA 80: 2632-2636
Schneider EM, Becker JU, Volkmann D (1981) Biochemical properties of potato phosphorylase change with its intracellular localization as revealed by immunological methods. Plata 151: 124-134
Shewry PR, Napier JA, Tatham AS (1995) Seed storage proteins: Structures and biosynthesis. Plant Cell 7: 945-956
Shutov AD, Vaintraub IA (1987) Degradation of storage proteins in germinating seeds. Phytochemistry 26(6): 1557-1566
Slade L, Levine H (1991) Beyond water activity: recent advances based on an alternative approach to the assesment of food quality and safety. Critical Reviews in Food Science and Nutrition 30 (2-3) : 115-360
Steup M (1988) Starch degradation. In: Press J (ed) Biochemistry of plants, vol 14, Carbohydrates. Academic Press, New York, pp 255-296
Storey R D (1986) Plant endopeptidases. In: Plant Proteolytic Enzymes, M. Dalling, ed (Boca Raton, FL: CRC Press), pp. 119-135
Storozhenko S, De Pauw P, Van Montagu M, Kushnir S (1998) The heat shock element is a functional component of the Arabidopsis APX1 gene promoter. Plant Physiol 116: 1005-1014
Tananuwong K, Reid DS (2004) DSC and NMR relaxation studies of starch-water interactions during gelatinization. Carbohydrate Polymers 58: 345-358
Törmäkangas K, Hadlington JL, Pimpl P, Hillmer S, Brandizzi F, Teeri TH, Denecke J (2001) A vacuolar sorting domain may also influence the way in which proteins leave the endoplasmic reticulum. Plant Cell 13: 2021-2032
Tornero P, Conejero V, Vera P (1996) Primary structure and expression of a pathogen-induced protease (PR-P69) in tomato plants: similarity of functional domains to subtilisin-like endoproteases. Proc Natl Acad Sci USA 93: 6332-6337
Toyo-Oka T (1982) Phosphorylation with cAMP dependent protein kinase renders bovine cardiac troponin sensitive to te degradation by calcium-activated neutral protease. Biochem Biophys Res Commun 107: 44-50
Vera P, Conejero V (1998) Pathogenesis-related proteins of tomato. P-69 as an alkaline endoproeinase. Plant Physiol 87: 58-63
Vierstra RD, Quail PH (1983) Purification and initial characterization of 124-kilodalton phytochrome from Avena. Biochemistry 22(10): 2498-2505
Vierstra RD (2003) The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins. Trends Plant Sci. 8: 135-142
von Wettstein D, Gough S, Kannangara CG (1995) Chlorophyll biosynthesis. Plant Cell 7: 1039-1057
Wilk S, Orlowski M (1980) Cation-sensitive neutral endopeptidase: isolation and specificity of the bovine pituitary enzyme. J Neurochem 35: 1172-1182
Wilk S, Orlowski M (1983) Evidence that pituitary cation-sensitive neutral endopeptidase is a multicatalytic protease complex. J Neurochem 40: 842-849
Wiśniewski K, Zagadańska B. (2001) Genotype-dependent proteolytic response of spring wheat to water deficiency. J Exp Bot. 52: 1455-1463
Wenzel T, Eckerskorn C, Lottspeich F, Baumeister W (1994) Existence of a molecular ruler in proteasomes suggested by analysis of degradation products. FEBS Lett 349: 205-209
Wu S-H, Wong C, Chen J, Lin B-C (1994). Isolation of a cDNA encoding a 70 kDa heat shock cognate protein expressed in the vegetative tissue of Arabidopsis. Plant Mol Biol 25: 577-583
Ye Z-H, Varner J (1993) Gene expression patterns associated with in vitro tracheary element formation in isolated single mesophyll eclls of Zinnia elegans. Plant Physiol 103: 805-813
Zeeman SC, Thorneycroft D, Schupp N, Chapple A, Weck M, Dunstan H, Haldimann P, Bechtold N, Smith AM, and Smith SM (2004) Plastidial a-Glucan Phosphorylase Is Not Required for Starch Degradation in Arabidopsis Leaves But Has a Role in the Tolerance of Abiotic Stress. Plant Physiology 135: 849-858