臺灣博碩士論文加值系統

摘 　　 要
為瞭解發芽綠豆子葉內VrPE-1(a putative Asparaginyl endopeptidase)的表現及其生理功能，利用VrPE-1的抗體及其對基質(Z-Ala-Ala-Asn-MCA)的活性來追蹤及純化發芽綠豆子葉內的VrPE-1。以免疫轉印分析發芽第0~7天綠豆子葉內VrPE-1以發芽第5天的訊號最強。發芽第5天綠豆根部及胚軸的VrPE-1及Asparaginyl peptidase活性強度都比綠豆子葉明顯。
收取發芽第五天綠豆子葉，以0~45％飽和度(NH4)2SO4分劃所萃取的蛋白質溶液通入Q-Sepharose column陰離子交換樹脂管柱層析，收集具有Asparaginyl peptidase活性部分通入下一Butyl-Sepharose 4 fast flow column疏水性樹脂管柱層析，作更進一步的純化。最後收集具有Asparaginyl peptidase活性部分作更進一步的生化特性分析。
由Butyl-Sepharose 4 管柱層析收集具有Asparaginyl peptidase活性部分，在SDS-PAGE上仍還有許多蛋白帶，雖然以Anti-VrPE-1抗體作西方墨點分析只有一明顯的免疫反應蛋白帶（~18 kDa），但無法確定此即為VrPE-1。以此部分作一些生化特性分析結果：最適貯存的酸鹼值為5.0。10 μM PMSF、1 μM Leupeptin及10 μM NEM對Asparaginyl endopeptidase有50% 的抑制效果；1 μM E-64只有些微抑制效果。

Abstract
To understand the physiological function of VrPE-1, a putative asparaginyl endopeptidase, in the cotyledons of germinated mung beans (Vigana radiate), a polyclonal antibody against VrPE-1 and an enzymatic activity of asparaginyl peptidase with Z-Ala-Ala-Asn-MCA as a substrate were utilized for the analysis of the expression and the purification of VrPE-1 from germinated mung beans. By Western blot analysis, VrPE-1 occurs in cotyledons, hypocotyls and roots, but not in leaves, of germinated mung beans on 5 DAI (days-after-imbibition), and its expression in cotyledons reaches a maximum on 5 DAI.
Ammonium sulfate fraction (0-45% saturation) of the crude protein extract from 5-DAI cotyledons was chromatographed on a Q-Sepharose column. The fractions exhibiting asparaginyl peptidase activity were pooled and further chromatographed on a butyl-Sepharose 4 column. The chromatogram of butyl-Sepharose 4 showed a single peak of asparaginyl peptidase activity.
The enzymatically active fractions were collected and pooled for further biochemical characterization. The resulting pooled fraction showed several bands on SDS-polyacrylamide gel and only one band with a molecular mass of 18 kDa on Western blot. The pooled fraction had storing pH optimum around 5.0 for the asparaginyl peptidase activity with Z-Ala-Ala-Asn-MCA as a substrate. The fraction showed about 50% enzymatic inhibition with 10 μM PMSF, 1 �嵱 leupeptin or 10 μM NEM, and small effect with 1 �嵱 E-64 inhibitor.

目 錄
中文摘要…………………………………………………………………4
英文摘要…………………………………………………………………5
壹、前言………………………………………………………………… 6
貳、文獻整理…………………………………………………………… 9
一、Asparaginyl endopeptidase的介紹……………………………… 9
二、Asparaginyl endopeptidase對植物的重要性……………………11
三、動物型Legumain與植物型Legumain的差異…………………12
參、材料與方法…………………………………………………………15
一、樣品的製備與處理………………………………………………15
（一）綠豆芽的培養…………………………………………………15
（二）綠豆芽酵素液的萃取…………………………………………15
（三）硫酸銨(NH4)2SO4分劃………………………………………16
（四）透析……………………………………………………………17
二、蛋白質定量………………………………………………………18
三、SDS-聚丙烯醯胺膠電泳………………………………………19
四、蛋白酶活性染色………………………………………………22
五、西方墨點分析（Western blot analysis）………………………25
六、Enzymatic activity assay…………………………………………27
七、純化………………………………………………………………28
（一）Q-Sepharose 陰離子交換樹脂管柱層析…………………28
（二）Butyl-Sepharose 4 Fast Flow column疏水性樹脂管柱層析 …………………………………………………………30
八、最適貯存酸鹼值…………………………………………………32
九、抑制劑之影響……………………………………………………33
肆、結果…………………………………………………………………35
一、發芽綠豆子葉酵素液的製備……………………………………35
二、發芽第5日綠豆各器官Asparaginyl peptidase活性的比較…36
三、硫酸銨分劃………………………………………………………37
四、Q-Sepharose管柱層析…………………………………………39
五、Butyl-Sepharose 4管柱層析…………………………………40
六、生化特性…………………………………………………………42
（一）最適貯存酸鹼值（pH）……………………………………42
（二）抑制劑的影響……………………………………………43
伍、討論………………………………………………………………44
一、綠豆子葉酵素液的製備………………………………………44
二、發芽第5天綠豆子葉、根、胚軸、葉子的活性分析………45
三、硫酸銨分劃……………………………………………………45
四、Q-Sepharose管注層析………………………………………46
五、Butyl-Sepharose 4管注層析………………………………47
六、生化特性………………………………………………………48
陸、參考文獻…………………………………………………………50
柒、圖與表……………………………………………………………57

陸、參考文獻
Abe, Y., Sbirane, K., Yokosawa, H., Mitta, M., Kato, I. and Ishii, S.（1993）Asparaginyl endopeptidase of jack bean seends. J. Biol.Chem. 268：3525-3529.
Ashton, F. M.（1976）Mobilization of storage proteins of seeds. Plant Physiol. 27：95-117.
Becker, C., Shutov, A. D., Nong, V. H., Senyuk, V. I., Jung, R., Horstmann, C., Fischer, J., Nielsen, N. C. and Muntz, K.（1995）Purification, cDNA cloning and characterization of proteinase B, an asparagines-specific endopeptidase from germinating vetch（Vicia sativa L.）seeds. Eur. J. Biochem. 228：456-462.
Baumgartener, B. and Chrispeels, M. J.（1997）Purification and characterization of vicilin peptidohydrolase, the major endopeptidase in the cotyledons of mung-bean seeding. Eur. J. Biochem. 77：223-233.
Catsimpoolas, N., Campbell, T. G. and Meyer, E. W.（1968）Immunochemical study of changes in reserve proteins of germinating soybean seeds. Plant Physiol. 43：799-805.
Csoma, C.and Polgar, L.（1984）Proteinase from germinating bean cotyledons. Biochem. J. 222：769-776.
Chen, J. M., Dando, P. M., Rawlings, N. D., Brown, M. A., Young, N. E., Stevens, R. A., Hewitt, E., Watts, C. and Barrett, A. J.（1997）Cloning, isolation, characterization of mammalian legumain, an asparaginyl endopeptidase. The Journal of Biological Chemistry. 272：8090-8098.
Cooper, T. G..（1976）The Tools of Biochemistry. Wiley-Interscience Publication. P：194-209.
Dando, P. M., Fortunato, M., Smith, L., Graham Knight, C. Mckendrick, J. E. and Barrett, A. J.（1999）Pig kidney legumain：an asparaginyl endopeptidase with restricted. Biochem. J. 339：743-749.
Garg, G. K. and Virupaksha, T. K.（1970）Acid Protease from germinated sorghum. Eur. J. Biochem. 17：4-12.
Higgins, T. J. V.（1984）Synthesis and regulation of major proteins in seeds. Plant Physiol. 35：191-221.
Hara-Nishimura, I., Inoue, k. and Nishimura, M.（1991）A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into the mature forms. Febs letters. 294：89-93.
Ishii, shin-ichi.（1994）Legumain：asparaginyl endopeptidase. Methods in enzymology. 244：604-615.
James, Michael N. G.. and Sielecki , A. R. （1986）Molecular structure of an aspartic proteinase zymogen, procine pepsinogen, at 1.8 A0 resolution. Nature. 319：33-39.
Johansen, H. T., Graham Knight, C. and Barrett, A. j.（1999）Colorimetric and fluorimetric microplate assays for legumain and a staining reaction for detection of the enzyme after electrophoresis. Analytical Biochemistry. 273：278-283.
Kuroyanagi, M., Nishimura, M. and Hara-Nishimura, I.（2002）Activation of Arabidopsis vacuolar processing enzyme by self catalytic removal of an auto-inhibitory domain of c-terminal propeptide. Plant Cell Physiol. 43：143-：151.
Kembhavi, A. A., Buttle, D.J., Knight, G.. and Barrett, A. J.（1993）The two cysteine endopeptidase of legume seeds：purification and characterization by use of specific fluorometric assays. Archives of biochemistry and biophysics. 303：208-213.
Kassell, B. and Kay, J.（1973） Zymogens of proteolytic enzymes. Science. 180：1022-1027.
Mitsuhashi, W., Komokazu, T. and Minamikawa, T.（1984）Influence of axis removal on amino-, carboxy- and endopeptidase activities in cotyledons of germinating Vigna mungo seeds. Plant and Cell Physiol.
Muntz, K. and Shutov, A. D.（2002）Legumains and their functions in plants. Plant Science. 10：1360-1385.
Mitsuhashi, W., Koskiba, T. and Minamikawa, T.（1986）Separation and characterization of two endopeptidase from cotyledons of germinating Vigna mungo seeds. Plant Physiol. 80：628-634.
Mitsuhashi, W., Koshiba, T. and Minamikawa, T.（1984）Micro-spot assay and detection on polyacrylamide gel of Mung bean endopeptidase activity by substrate- polyacrylamide gel plate method. Agric. Biol. Chem. 48：2387-2388.
Muntz, k., Belozersky, M. k. Dunaevsky, Y. E. Schlereth, A. and Tiedemann , J.（2001）Stored proteinase and the initiation of storage protein mobilization in seeds during germination and seeding growth. Journal of experimental botany. 52：1714-1752.
Mitsuhashi, W. and Minamikawa, T.（1989）Synthesis and posttransalational activation of sulfhydryl-endopeptidase in cotyledpns of cerminating Vigna mungo Seeds. Plant Physio. 89：274-279.
Okamoto, T. and minamikawa, T.（1995） Purification of a processing enzyme（VmPE-1）that is involved in post-translational processing of vacuolar cysteine proteinase（SH-EP）. Eur. J. Biochem. 231：300-305.
Oakley, B. R., Kirsch, D. R. and Ronald Morris, N.（1980）A simplified ultrasenitive silver stain for detecting proteins in polyacrylamide gels. Analyticial Biochemistry. 105：361-363.
Okamoto, T., Yuki, A., Mitsuhashi, N. and Mimamikawa, T.（1999） Asparaginyl endopeptidase（VmPE-1）and autocatalytic processing synergistically activate the vacuolar cysteine proteinase（SH-EP）. Eur. J. Biochem. 264：223-232.
Okamoto, T. and minamikawa, T.（1999）Molecular cloning and characterization of Vigna mungo processing enzyme 1（VmPE-1）, an asparaginyl endopeptidase possibly involved in post-translational processing of vacuolar cysteine proteinase（SH-EP）. Plant Molecular Biology. 39：63-73.
Rotari, V. I., Dando, P. M. and Barrett, A. J.（2001）Legumain forms from plants and animals differ in their specificity. Biol. Chem. 382：953-959.
Scopes, R. K.（1994）Protein Purification. Springer-Verlag New York Publication. P：293-307.
Sutoh, K., Kato, H.and Minamikawa, T.（1999）Indentification and possible roles of three types of endopeptidase from germinated wheat seeds. J. Biochem. 126：700-707.
Shimada, T., Yamada, K., Kataoka, M., Nakaune, S., Koumoto, Y., Kuroyanagi, M., Tabata, S., Kato, T., Shinozaki., Seki, M., Kobayashi, M., Kondo, M., Nishimura, M. and Hara-Nishimura, I.（2003）Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana. The Journal of Biological Chemistry. 278：32292-32299.
Senyuk, V., Rotari, V., Becker, C., Zakharov, A. and Horstmann, C. （1998）Does an asparaginyl-specific cysteine endopeptidase trigger phaseolin degradation in cotyledons of kidney bean seedlings？ Eur. J. Biochem. 258：546-558.
Sadeghipour, H. R. and Bhatla, S. C.（2002）Differential sensitivity of oleosins to proteolysis during oil body mobilization in sunflower seedings. Plant Cell Physiol. 43：1117-1126.
Schwarz, G.., Brandenburg, J., Reich, M., Burster, T., Driessen, C. and Kalbacher, H.（2002）Characterization of legumain. Biol. Chem. 383：1813-1816.
Chrispeels, M. J., Baumgartner, B. and Harris, N.（1976）Regulation of reserve protein metabolism in the cotyledons of mung bean seedlings. Proc. Natl. Acad. Sci. USA. 73：3168-3172.
Harris, N. and Chrispeels, M. J.（1975）Histochemical and biochemical observations on storage protein metabolism and protein body autolysis in cotyledons of germinating mung bean. Plant Physiol. 56：292-299.
Minamikawa, T. and Okamoto, T.（1998）A vacuolar cysteine endopeptidase （SH-EP）that digests seed storage globulin：characterization, regulation of gene expression, and posttranslational processing. J. Plant Physiol. 152：675-682.
Chrispeels, M. J. and Boulter , D.（1975）Control of storage protein metabolism in the cotyledons of germinating mung beans：role of endopeptidase. Plant Physiol. 55：1031-1037.
Okamoto, T., Nakayama, H., Seta, K. Isobe, T. and Minamikawa, T. （1994）Posttranslational processing of a carboxy-terminal propeptide containing a KDEL sequence of plant vacuolar cysteine endopeptidase（SH-EP）. FEBS letters. 351：31-34.
Ryan, C. A. （1973）Proteolytic enzymes and their inhibitors in plants. Plant Physiol. 24：173-196.
Tiedemann, J., Schlereth, A. and Muntz, K.（2001）Differential tissue-specific expression of cysteine proteinases forms the basis for the fine-tuned mobilization of storage globulin during and after germination in legumglobulin during and after germination in legume seeds. Planta. 212：728-738.
劉慶鎮（2004）綠豆老化相關半胱胺酸蛋白酶VrCys2的表現、純化與鑑定。國立台灣海洋大學生物科技研究所碩士學位論文。
Yang, Wei-Horng. and Chen, Ching-San.（2002）Genes and enzymes involved in plant senescence：Senescence in mung bean. Institute of Botany Academia Sinica.（未發表）