臺灣博碩士論文加值系統

蔗糖磷酯合成■（ sucrose phosphate synthase , EC 2.4.1.14，簡稱SPS）是高等植物中蔗糖生合成的重要酵素。將台農57號甘藷塊根所誘導出的懸浮細胞，培養於含高濃度山梨醣醇的高滲透逆境培養基中，以此作為實驗材料。經過陰離子交換管柱（DEAE-Sephacel）、PEG沉澱、親和管柱（■-aminohexyl Sepharose 4B ）及快速蛋白質分離管柱（FPLC - Mono Q）等層析法後，得到純化倍率43.8倍，回收率3.3 ﹪之部分純化SPS。以原態-PAGE及SDS-PAGE，推測其原態分子量約為540 kD，次單元體分子量約為130 kD。
在生化性質方面，高滲透逆境下甘藷懸浮細胞SPS最適反應溫度為37 ℃，0 ~ 37 ℃可保持80 ﹪以上的活性。酵素最適反應pH值為7.5，在pH值6.5 ~ 7.5 之間酵素最穩定。鎂離子會促進酵素活性，在10 mM時達到最大，而汞離子則會抑制其活性。酵素對其基質果醣 6-磷酯與UDPG之Km值分別為5.15 mM 及47.8 mM。
在效應物之調控機制方面，高滲透逆境下甘藷懸浮細胞SPS的活性不會被葡萄糖 6-磷酯所促進，亦不會被無機磷酸鹽所抑制。此與光合組織者不同，而近似於甘藷塊根與癒創組織。

Sucrose phosphate synthase（SPS；EC 2.4.1.14）has been partially purified 43.8-fold from an extract of induced suspension cells of sweet potato (Ipomoea batatas , L. Lam.) tuberous root , which has been incubated and subcultured in osmotic high-stressed medium with 0.6 M sorbitol . SPS was successively purified by DEAE-Sephacel ion exchange , PEG precipitation , ■-aminohexyl Sepharose 4B and FPLC—Mono Q chromatography. The subunit and native molecular mass of SPS were estimated about 130 and 540 kD , respectively , by native- and SDS-PAGE methods , respectively .
The optimal temperature for SPS enzyme activity is 37 ℃ while the enzyme remain stable from 0 ℃ to 37 ℃. The optimal pH for SPS was 7.5 , and between pH 6.5 ~7.5 , the SPS maintained about 80 ﹪of the maximum activity. The enzyme was activated by Mg2+ and inhibited by Hg2+ , while it was not affected apparently by Mn2+ and Ca2+ . The Km values for F-6-P and UDPG are 5.15 and 47.8 mM , respectively .
SPS from suspension cells of sweet potato treated with high osmotic stress was not activated by G-6-P . The enzyme was not inhibited by inorganic phosphate either . It behaves different from the SPS of photosynthetic tissues but similiar to the SPS from tuberous roots .

第一章 緒論 1
第一節 蔗糖在生物體內的生理功能 1
第二節 蔗糖磷酯合成■ 2
第三節 蔗糖磷酯合成■活性之調控 3
第四節 不同植物間蔗糖磷酯合成■之比較6
第五節 蔗糖磷酯合成■可能出現複合體 7
第六節 蔗糖磷酯合成■之其他研究 7
第二章 材料與方法 10
第一節 材料、藥品與儀器 10
第二節 蛋白質定量法 12
第三節 蔗糖磷酯合成■活性測定 13
第四節 蔗糖合成■之酵素活性分析 15
第五節 電泳檢定系統 16
5.1原態膠體電泳 16
5.2 SDS-膠體電泳 20
5.3 膠體染色法 22
5.3.1 CBR 染色法 22
5.3.2 膠片乾燥法 23
第六節 性質檢定 23
第七節 蔗糖磷酯合成■之純化 25
7.1 酵素的粗抽及PEG分劃 25
7.2 ■-aminohexyl Sepharose 4B 26
7.3快速蛋白質液相層析 28
第八節 酵素生化性質分析 30
8.1 最適反應溫度 31
8.2 熱安定性 31
8.3 最適 pH 值測定 31
8.4 pH 安定性 31
8.5 G-6-P 濃度對酵素活性的影響 32
8.6 Pi 濃度對酵素活性的影響 32
8.7 二價金屬離子對酵素活性的影響 32
第三章 結果與討論 33
第一節 材料的選用 33
第二節 酵素純化策略的探討 38
第三節 酵素純化的結果 39
第四節 高滲透逆境甘藷懸浮細胞中蔗糖磷酯合成■的
生化性質分析 52
第四章 結論與展望 63
參考文獻 65

ap Rees, T. （1984）Sucrose metabolism. In storage carbohydrates in vascular plants. pp. 53-57 Cambridge University Press. London.
Pontis, H.G. （1978）On the scent of riddle of sucrose. Trend in Biochem. Sci., 3：137-139.
Singh, M. B., Knox, R. B. （1984）Invertases of Lilium pollen. Characterization and activity during in vitro germination. Plant Physiol. 74：510-515.
Huber, S. C., Huber, J. L., (1996) Role and regulation of sucrose-phosphate synthase in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 17：431-444
Cardini, C. E., Leloir, L. F., Chiriboga, J. （1955）The biosynthesis of sucrose J. Biol. Chem. 214：149-154.
Salvucci, M. E., Drake, R. R., Haley, B. B.,(1990) Purification and photoaffinity labeling of sucrose phosphate synthase from spinach leaves. Arch. Biochem. Biophys. 281：212-218
Lunn, J. E., Ap,-Rees, T.,(1990) Purification and properties of sucrose phosphate synthase from seeds of Pisum sativum. Phytochemistry. 29：1057-1063
Bruneau, J. M., Worrell, A. C., Cambou, B., Lando, D., Voelker, T. A., (1991) Sucrose phosphate synthase, a key enzyme for sucrose biosynthesis in plants. Protein purification from corn leaves and immunological detection. Plant Physiol. 96：473-478
Reimholz, R., Geigenberger, P., Stitt, M.,(1994) Sucrose phosphate synthase is regulated via metabolites and protein phosphorylation in potato tubers, in a manner analogous to the enzyme in leaves. Planta 192 ：480-488.
Hatzfeld, Wolf-D., Moesinger, E.,(1995) Active-site-directed inhibition of sucrose-phosphate synthase by Cibacron blue F3G-A and 1-deoxynojirimycin. Planta 197：296-230
Walker, J, L., Huber, S. C.,(1989) Regulation of sucrose-phosphate-synthases activity in spinach leaves by protein level and covalent modification. Planta 177：116-120
Douglas, C. D., Huber, S. C., (1983) Regulation of spinach leaf sucrose phosphate synthase by glucose-6-phosphate, inorganic phosphate, and pH. Plant Physiol 73 ：989-994
Huber, S. C., Rufty, T. W., Keer, P. S., (1984) Effect of photoperiod on photosynthesis partitioning and diurnal rhythms in sucrose phosphate synthase activity in leaves of soybean and tobacco. Plant Physiol. 75 ：1080-1084
Stitt, M., Wilke, I., Feil, R., Heldt, H. W., (1988) Coarse control of sucrose -phosphate synthase in leaves . Planta 174：217-230
Huber, J. l. A., Huber, S.C., Nielsen, T. H.,(1989) Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity. Arch. Biochem. Biophy. 270： 681-690
Siegl, G., Stitt, M., (1990) Partial purification of two forms of spinach sucrose-phosphate synthase which differ in their kinetic properties. Plant Sci. 66：205-210
McMichael. R. W. Jr., Klein. R. R., Salvucci. M.. E., Huber. S. C, （1993）Identification of the major regulation phosphorylation site in sucrose phosphate synthase. Arch. Biochem. Biophys. 307：248-252.
Huber. S. C., Huber. J. L. A., （1992）Role of sucrose-phosphate synthase in sucrose metabolism in leaves. Plant Physiol. 99：1275-1278.
Huber. S. C. Huber. J. L., （1991）In vitro phosphorylation and inactivation of spinach leaf sucrose-phosphate synthase by an endogenous protein kinase. Biochim. Biophts. Acta. 1091：393-400.
McMichael. R. W. Jr., Bachman. M., Huber. S. C., （1995）Spinach leaf sucrose-phosphate synthase and nitrate reductase are phosphorylated/inactivated by multiple protein kinase in vitro. Plant Physiol. 108：1077-1082.
Weiner, H., McMichael, R. W. Jr., Huber, S. C., (1992) Identification of factors regulating the phosphorylation status of sucrose-phosphate synthase in vivo. Plant Physiol. 99：1435-1442
Huber, S. C., Nielsen, T. H., Huber, J. L. A., Pharr, D. M.,(1989) Variation among species in light activation of sucrose-phosphate synthase. Plant Cell Physiol. 30：277-285
Galtier, Foyer. C. H., Huber, J. L. A.(1993) Effects of elevated sucrose-phosphate synthase activity on photosynthesis, assimilate partitioning and growth in tomato (Lycopersicon esculentum var. UC 82B). Plant Physiol. 101：535-543
Kleim, R. R., Crafts-Brandner, S. J., Salvucci, M. E. （1993）Identification of uridine binding domain of sucrose-phosphate synthase . Cloning and expression of a region of the protein that photoaffinity labels with 5-azidouridine diphosphate-glucose. Plant Physiol. 102：529-536
Worrell, A. C., Bruneau, J. M., Summerfelt, K., Boersig, M., Voelker, T. A. （1991）Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning. Plant Cell 3：1121-1130
Hesse, H., Sonnewald, U., Willmitzer, L., (1995) Cloning and expression analysis of sucrose-phosphate synthase from sugar beet ( Beta vulgaris L. ). Mol. Gen. Genet.247：515-520
Zrenner, R., Salanoubat, M., Willmitzer, L., Sonnewald, U., (1995) Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants ( Solanum tuberosum L. ) Plant J. 7：97-107
Valdez-Alarcon, J. J., Ferrando, M., Salerno, G., Jimenez-Moraila, B., Herrera-Hstrella, L.,(1996) Characterization of a rice sucrose-phosphate synthase -encoding gene. Gene. 170：217-222
Galtier,N., Foyer. C. H.,Murchie, E., Alred, R., Quick, P., et al.(1995) Effects of light and atmospheric carbon dioxide enrichment on photosynthesis and carbon partitioning in the leaves of tomato ( Lycopersicon esculentum L.) plant over-expressing sucrose phosphate synthase.. J. Exp. Bot.46：1335-1344
Micallef, B. F., Haskin, K. A., Vanderveer, P. J., Roth, K. S., Shewmaker, C. K., Sharkey, T. D. （1995）Altered photosynthesis, flowering and fruiting in transgenic tomato plants that have an increased capacity for sucrose synthesis. Planta 196：327-334
Stitt, M., Sonnewald, U. （1995）Regulation of metabolism in transgenic plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46：341-368
Guy, C. L., Huber, J. L. A., Huber, S. C., (1992) Sucrose phosphate synthase and sucrose accumulating at low temperature. Plant Physiol. 100：502 ~ 503
Hurry, V. M., Malmberg, G., Gardestrom, P., Oquist, G., (1994) Effects of a short-term shift to low temperature and of long-term cold hardening on photosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase and sucrose phosphate synthase activity in leaves of winter rye ( Secale cereale L. ) Plant Physiol. 106：983 ~ 990
Cheink, N., Brenner, M. L., (1992) Effect of dark and light conditions and role of gibberellins and abscisic acid. Plant Physiol. 100：1230 ~ 1237
Cheikh, N., Brenner, M. L., Huber, J. L., Huber, S. C., (1992) Regulation of sucrose phosphate synthase by gibberellins in soybean and spinach plants. Plant Physiol. 100：1238 ~ 1242
Vassey, T. L., Sherkey, T. D., (1989) Mild water stress of Phaseolus vulgaris plants leads to reduced starch synthesis and extractable sucrose phosphate synthase activity. Plant Physiol. 89：1066 ~ 1070
Castrillo, M., (1992) Sucrose metabolism in bean plants under water deficit. J. Exp. Bot. 43：1557 ~ 1561
Amir, J., Preiss, J.（1982）Kinetic characterization of spinach leaf sucrose-phosphate synthase. Plant Physiol. 69：1027-1030
Murata, T. (1971) Enzymic mechanism of starch synthesis in sweet potato roots. Part IV. Sucrose synthetase of sweet potato roots. Nippon Nogei Kagaku Kaishi 45：441-448
Walker, J. L. A., Huber, S. C.（1989）Purification and preliminary characterization of sucrose-phosphate synthase using monoclonal antibodies. Plant Physiol. 89：518-524
Sonnewald,U., Quick, W. P., MacRae, E., Krause, K. P.,Stitt,（1993）Purification cloning and expression of spinach leaf sucrose phosphate synthase in E.coli. Planta 189：174-181
Dikran Toroser and Steven C. Huber , （1997）Protein phosphorylation as a mechanism for osmotic-stress activation of sucrose-phosphate synthase in spinach leaves . Plant Physiol . 114：947-955
Alok Krishna Sinha , Uday Vasudeo Pathre , Prafullachandra（1999）
Essential histidyl residues at the active site(s）of sucrose phosphate
synthase from Prosopis juliflora . Biochimica et Biophysica Acta 1388：397-404
Pontis, H. G. : Riddle of sucrose. In : Plant Biochemistry II, Vol. 13, pp. 79-117, Northcote, D.h., ED. Baltimore: University Park Press 1977
莊榮輝（1985）水稻蔗糖合成之研究。博士論文，國立台灣大學農 業化學研究所
鄭宇哲（1997）水稻乳熟期穀實蔗糖磷酯合成之生化研究。碩士
論文。國立台灣大學農業化學研究所
陳韋良（1998）甘藷塊根蔗糖磷酯合成之生化學研究。碩士論文。
國立台灣大學農業化學研究所
王恆隆（1999）滲透逆境誘發甘藷懸浮細胞累積澱粉之研究。博士
論文，國立台灣大學農業化學研究所
李木和（1999）甘藷癒創組織以光照及滲透逆境誘導蔗糖磷酯合
成ap Rees, T. （1984）Sucrose metabolism. In storage carbohydrates in vascular plants. pp. 53-57 Cambridge University Press. London.
Pontis, H.G. （1978）On the scent of riddle of sucrose. Trend in Biochem. Sci., 3：137-139.
Singh, M. B., Knox, R. B. （1984）Invertases of Lilium pollen. Characterization and activity during in vitro germination. Plant Physiol. 74：510-515.
Huber, S. C., Huber, J. L., (1996) Role and regulation of sucrose-phosphate synthase in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 17：431-444
Cardini, C. E., Leloir, L. F., Chiriboga, J. （1955）The biosynthesis of sucrose J. Biol. Chem. 214：149-154.
Salvucci, M. E., Drake, R. R., Haley, B. B.,(1990) Purification and photoaffinity labeling of sucrose phosphate synthase from spinach leaves. Arch. Biochem. Biophys. 281：212-218
Lunn, J. E., Ap,-Rees, T.,(1990) Purification and properties of sucrose phosphate synthase from seeds of Pisum sativum. Phytochemistry. 29：1057-1063
Bruneau, J. M., Worrell, A. C., Cambou, B., Lando, D., Voelker, T. A., (1991) Sucrose phosphate synthase, a key enzyme for sucrose biosynthesis in plants. Protein purification from corn leaves and immunological detection. Plant Physiol. 96：473-478
Reimholz, R., Geigenberger, P., Stitt, M.,(1994) Sucrose phosphate synthase is regulated via metabolites and protein phosphorylation in potato tubers, in a manner analogous to the enzyme in leaves. Planta 192 ：480-488.
Hatzfeld, Wolf-D., Moesinger, E.,(1995) Active-site-directed inhibition of sucrose-phosphate synthase by Cibacron blue F3G-A and 1-deoxynojirimycin. Planta 197：296-230
Walker, J, L., Huber, S. C.,(1989) Regulation of sucrose-phosphate-synthases activity in spinach leaves by protein level and covalent modification. Planta 177：116-120
Douglas, C. D., Huber, S. C., (1983) Regulation of spinach leaf sucrose phosphate synthase by glucose-6-phosphate, inorganic phosphate, and pH. Plant Physiol 73 ：989-994
Huber, S. C., Rufty, T. W., Keer, P. S., (1984) Effect of photoperiod on photosynthesis partitioning and diurnal rhythms in sucrose phosphate synthase activity in leaves of soybean and tobacco. Plant Physiol. 75 ：1080-1084
Stitt, M., Wilke, I., Feil, R., Heldt, H. W., (1988) Coarse control of sucrose -phosphate synthase in leaves . Planta 174：217-230
Huber, J. l. A., Huber, S.C., Nielsen, T. H.,(1989) Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity. Arch. Biochem. Biophy. 270： 681-690
Siegl, G., Stitt, M., (1990) Partial purification of two forms of spinach sucrose-phosphate synthase which differ in their kinetic properties. Plant Sci. 66：205-210
McMichael. R. W. Jr., Klein. R. R., Salvucci. M.. E., Huber. S. C, （1993）Identification of the major regulation phosphorylation site in sucrose phosphate synthase. Arch. Biochem. Biophys. 307：248-252.
Huber. S. C., Huber. J. L. A., （1992）Role of sucrose-phosphate synthase in sucrose metabolism in leaves. Plant Physiol. 99：1275-1278.
Huber. S. C. Huber. J. L., （1991）In vitro phosphorylation and inactivation of spinach leaf sucrose-phosphate synthase by an endogenous protein kinase. Biochim. Biophts. Acta. 1091：393-400.
McMichael. R. W. Jr., Bachman. M., Huber. S. C., （1995）Spinach leaf sucrose-phosphate synthase and nitrate reductase are phosphorylated/inactivated by multiple protein kinase in vitro. Plant Physiol. 108：1077-1082.
Weiner, H., McMichael, R. W. Jr., Huber, S. C., (1992) Identification of factors regulating the phosphorylation status of sucrose-phosphate synthase in vivo. Plant Physiol. 99：1435-1442
Huber, S. C., Nielsen, T. H., Huber, J. L. A., Pharr, D. M.,(1989) Variation among species in light activation of sucrose-phosphate synthase. Plant Cell Physiol. 30：277-285
Galtier, Foyer. C. H., Huber, J. L. A.(1993) Effects of elevated sucrose-phosphate synthase activity on photosynthesis, assimilate partitioning and growth in tomato (Lycopersicon esculentum var. UC 82B). Plant Physiol. 101：535-543
Kleim, R. R., Crafts-Brandner, S. J., Salvucci, M. E. （1993）Identification of uridine binding domain of sucrose-phosphate synthase . Cloning and expression of a region of the protein that photoaffinity labels with 5-azidouridine diphosphate-glucose. Plant Physiol. 102：529-536
Worrell, A. C., Bruneau, J. M., Summerfelt, K., Boersig, M., Voelker, T. A. （1991）Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning. Plant Cell 3：1121-1130
Hesse, H., Sonnewald, U., Willmitzer, L., (1995) Cloning and expression analysis of sucrose-phosphate synthase from sugar beet ( Beta vulgaris L. ). Mol. Gen. Genet.247：515-520
Zrenner, R., Salanoubat, M., Willmitzer, L., Sonnewald, U., (1995) Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants ( Solanum tuberosum L. ) Plant J. 7：97-107
Valdez-Alarcon, J. J., Ferrando, M., Salerno, G., Jimenez-Moraila, B., Herrera-Hstrella, L.,(1996) Characterization of a rice sucrose-phosphate synthase -encoding gene. Gene. 170：217-222
Galtier,N., Foyer. C. H.,Murchie, E., Alred, R., Quick, P., et al.(1995) Effects of light and atmospheric carbon dioxide enrichment on photosynthesis and carbon partitioning in the leaves of tomato ( Lycopersicon esculentum L.) plant over-expressing sucrose phosphate synthase.. J. Exp. Bot.46：1335-1344
Micallef, B. F., Haskin, K. A., Vanderveer, P. J., Roth, K. S., Shewmaker, C. K., Sharkey, T. D. （1995）Altered photosynthesis, flowering and fruiting in transgenic tomato plants that have an increased capacity for sucrose synthesis. Planta 196：327-334
Stitt, M., Sonnewald, U. （1995）Regulation of metabolism in transgenic plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46：341-368
Guy, C. L., Huber, J. L. A., Huber, S. C., (1992) Sucrose phosphate synthase and sucrose accumulating at low temperature. Plant Physiol. 100：502 ~ 503
Hurry, V. M., Malmberg, G., Gardestrom, P., Oquist, G., (1994) Effects of a short-term shift to low temperature and of long-term cold hardening on photosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase and sucrose phosphate synthase activity in leaves of winter rye ( Secale cereale L. ) Plant Physiol. 106：983 ~ 990
Cheink, N., Brenner, M. L., (1992) Effect of dark and light conditions and role of gibberellins and abscisic acid. Plant Physiol. 100：1230 ~ 1237
Cheikh, N., Brenner, M. L., Huber, J. L., Huber, S. C., (1992) Regulation of sucrose phosphate synthase by gibberellins in soybean and spinach plants. Plant Physiol. 100：1238 ~ 1242
Vassey, T. L., Sherkey, T. D., (1989) Mild water stress of Phaseolus vulgaris plants leads to reduced starch synthesis and extractable sucrose phosphate synthase activity. Plant Physiol. 89：1066 ~ 1070
Castrillo, M., (1992) Sucrose metabolism in bean plants under water deficit. J. Exp. Bot. 43：1557 ~ 1561
Amir, J., Preiss, J.（1982）Kinetic characterization of spinach leaf sucrose-phosphate synthase. Plant Physiol. 69：1027-1030
Murata, T. (1971) Enzymic mechanism of starch synthesis in sweet potato roots. Part IV. Sucrose synthetase of sweet potato roots. Nippon Nogei Kagaku Kaishi 45：441-448
Walker, J. L. A., Huber, S. C.（1989）Purification and preliminary characterization of sucrose-phosphate synthase using monoclonal antibodies. Plant Physiol. 89：518-524
Sonnewald,U., Quick, W. P., MacRae, E., Krause, K. P.,Stitt,（1993）
Purification cloning and expression of spinach leaf sucrose phosphate
synthase in E.coli. Planta 189：174-181
Dikran Toroser and Steven C. Huber , （1997）Protein phosphorylation
as a mechanism for osmotic-stress activation of sucrose-phosphate
synthase in spinach leaves . Plant Physiol . 114：947-955
Alok Krishna Sinha , Uday Vasudeo Pathre , Prafullachandra（1999）
Essential histidyl residues at the active site(s）of sucrose phosphate
synthase from Prosopis juliflora . Biochimica et Biophysica Acta
1388：397-404
Pontis, H. G. : Riddle of sucrose. In : Plant Biochemistry II, Vol. 13,
pp. 79-117, Northcote, D.h., ED. Baltimore: University Park Press
1977
莊榮輝（1985）水稻蔗糖合成之研究。博士論文，國立台灣大學農 業化學研究所
鄭宇哲（1997）水稻乳熟期穀實蔗糖磷酯合成之生化研究。碩士
論文。國立台灣大學農業化學研究所
陳韋良（1998）甘藷塊根蔗糖磷酯合成之生化學研究。碩士論文。
國立台灣大學農業化學研究所
王恆隆（1999）滲透逆境誘發甘藷懸浮細胞累積澱粉之研究。博士
論文，國立台灣大學農業化學研究所
李木和（1999）甘藷癒創組織以光照及滲透逆境誘導蔗糖磷酯合
成之表現與其生化性質探討。碩士論文。 國立台灣大學農業化
學研究所
張秋金（1999）水稻白化苗蔗糖磷酯合成之生化學研究。碩士論
文。國立台灣大學農業化學研究所
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