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研究生(外文):Yi-Lung Hsu
論文名稱(外文):Purification and Characterization of Histidine Tagged Vacuolar H+ -pyrophosphatase Expressed in Yeast
指導教授(外文):Rong-Long Pan
外文關鍵詞:V-PPasehis-taggedpurificationheterologous expressionyeastSDS-PAGEintrinsic absorptionintrinsic fluorescence
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液泡無機焦磷酸水解酵素,主要存在於高等植物和一些細菌中,利用水解PPi 提供二級主動運輸系統所需的能量。在真核生物或原核生物的有機體中,蛋白質包含六個連續的組胺酸,可輕易地使用金屬親和性管柱層析,把其純化出來。利用此方法所得到的蛋白質,是高純度且量又多,所以可用來做結構的研究和抗體的製備。在我們的實驗中,我們第一次成功地將異體表現的液泡無機焦磷酸水解酵素由酵母菌純化出來,就是利用此金屬親和性管柱層析(Ni2+-NTA)。藉由硫酸十二脂鈉-聚丙烯醯胺凝膠電泳法(SDS-PAGE) 和西方轉漬(Western blot) 的分析,我們可以得到液泡無機焦磷酸水解酵素的分子量約為73 kDa。
我們再將液泡無機焦磷酸水解酵素作一些特性分析的實驗,藉由酵素動力學的實驗,可得知Km為100 uM和Vmax為111 umol PPi mg-1h-1。而酵素活性,在其基質(Mg2+/ PPi) 比例等於1:1且pH值等於8的時候,酵素有最大活性。綜合所有實驗所得到的結果,我們可以發現在異體表現的液泡無機焦磷酸水解酵素和植物原生之液泡無機焦磷酸水解酵素,在其性質上是相似的,所以這證明液體表現的方法是可行的。另外,利用自身吸收(intrinsic absorption)和自身螢光(intrinsic fluorescence)光譜,所得到的結果,只能初步了解蛋白質與鉀離子和鈣離子作用時,在構形上會有所改變,未來可作一些輔以同位素(isotope)、 雙圓旋光儀(circular dichroism)等實驗,進一步了解蛋白質與鉀離子和鈣離子的結合機制。

H+-PPase (E.C. existed primarily in higher plants and several bacteria and requires PPi as energy source, supplying energy to the secondary active transport system. Fusion proteins containing a string of his-tag made it easy to be purified from both eukaryotic and prokaryotic organisms. The high purity and large-scale yield were useful for structural studies and immunogenic peptides preparation. In the present study, we first successfully purified vacuolar H+-pyrophosphatase heterologously expressed in yeast microsome. The technique of purification we used is immobilized-metal affinity chromatography, Ni2+-NTA (Nitrilotriacetic acid). Analysis with the SDS-PAGE (sodium dodecyl sulfate- polyacrylamine gel electrophoresis) and the Western blot of purified his-tagged vacuolar H+-PPase showed that only one single polypeptide with molecular mass of about 73 kDa was obtained.
After analyzing enzymatic kinetics, we obtained a Km of 100 uM and Vmax of 111 umol PPi mg-1h-1. The maximum enzymatic activity was measured with the substrate of Mg2+/ PPi ratio of 1:1 at pH 8.0. Taken together, the main properties of vacuolar H+-PPase heterologously expressed were similar to the endogenous one from mung bean seedlings. The results demonstrate the method of heterologous expression of V-PPase is feasible. By the intrinsic spectroscopy, we only knew that K+ and Ca2+ bind to V-PPase resulting in conformational changes. Further work is to investigate the mechanism of binding K+ and Ca2+ to V-PPase by using isotope, CD, and so on.

Table of Content
Abstract in Chinese-------------------------------------3
Abstract----------------------------------------------- 5
Introduct---------------------------------------------- 7
Material and Methods---------------------------------- 14
Results----------------------------------------------- 23
References-------------------------------------------- 36
Table and Figures -------------------------------------42

1. Baykov, A. A., Bakaleva, N. P., and Rea, P. A. (1993) Steady-state kinetics of substrate hydrolysis by vacuolar H+-pyrophosphatase. A simple three state model. Eur. J. Biochem. 217, 755-762.
2. Bradford, M. (1976) A rapid and sensitive method for the quantities of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248-254.
3. Darley, C. P., Skiera, L. A., Northrop, F. D., Sanders, D., and Davies, J. M. (1998) Tonoplast inorganic pyrophosphatase in Vicia faba guard cells. Planta 206, 272-277.
4. Gordon-Weeks, R., Steele, S. H., and Leigh, R. A. (1996) The role of magnesium, pyrophosphate, and their complexes as substrate and activators of the vacuolar H+-pumping inorganic pyrophosphatase. Plant Physiol. 111, 195-202.
5. Hung, S. H. (1997) Molecular analysis and heterologous expression of vacuolar H+-translocating enzymes. Doctoral thesis, Institute of Radiation Biology, National Tsing Hua University, Taiwan.
6. Von Jagow, G., Link, T. A., and Schagger, H (1994) Purification strategies for membrane proteins. A Practical Guide to Membrane Protein Purification. Von Jagow, G., and Schagger, H, ed. Academic Press, California, pp. 1-21.
7. Kim, E. J., Zhen, R. G., and Rea, P. A. (1995) Site-directed mutagenesis of vacuolar H+-pyrophosphatase. Necessity of Cys634 for inhibition by maleimides but not catalysis. J. Biol. Chem. 270, 2630-2635.
8. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
9. Lakowicz, J. R. (1999) Principles of fluorescence spectroscopy. 2nd edition. Kluwer Academic/Plenum Publishers, New York, pp. 15-63
10. Lanfermeijer, F. C., Venema, K., and Palmgren, M. G. (1998) Purification of a histidine-tagged plant plasma membrane H+-ATPase expressed in yeast. Protein Expr. Purif. 12, 29-37
11. Leigh, R. A., Pope, A. J., Jennings, I. R., and Sanders, D. (1992) Kinetics of the vacuole proton pyrophosphatase. Plant Physiol. 100, 1698-1705.
12. Leigh, R. A. (1997) Solute composition of vacuoles. In Advances in Botanical Research, Vol. 25: The Plant Vacuole, R. A. Leigh and D. Sanders, eds. Academic Press, London, pp. 171-194.
13. Maeshima, M., and Yoshida, S. (1989) Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean. J. Biol. Chem. 264, 20068-20073.
14. Maeshima, M. (1990) Oligomeric structure of H+-translocating inorganic pyrophosphatase of plant vacuoles. Biochem. Biophys. Res. Commun. 168, 1157-1162.
15. Maeshima, M. (1991) H+-translocating inorganic pyrophosphatase of plant vacuoles. Inhibition by Ca2+, stabilization by Mg2+ and immunological comparison with other inorganic pyrophosphatases. Eur. J. Biochem. 196, 11-7.
16. Maeshima, M. (2000) Vacuolar H+-pyrophosphatase. Biochim. Biophys. Acta 1465, 37-51.
17. Marty, F. (1999) Plant vacuoles. Plant Cell 11, 587-599.
18. Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258, 598-599
19. Rea, P. A., Kim, Y., Sarafian, V., Poole, R. J., Davies, J. M., and Sanders, D. (1992) Vacuolar H+-translocating pyrophosphatase: a new category of ion translocase. Trends Biochem. Sci. 17, 348-353.
20. Rea, P. A. and Poole, R. J. (1993) Vacuolar H+-translocating pyrophosphatase. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 157-180.
21. Sato, M. H., Maeshima, M., Ohsumi, Y., and Yoshida, M. (1991) Dimeric structure of H+-translocating pyrophosphatase from pumpkin vacuolar membranes. FEBS Lett. 290, 177-180.
22. Sulkowski, E. (1985) Purification of proteins by IMAC. Trends Biotechnol. 3, 1-7.
23. Tzeng, C. M., Yang, C. Y., Yang, S. J., Jiang, S. S., Kuo, S. Y., Hung, S. H., Ma, J. T., and Pan, R. L. (1996) Subunit structure of vacuolar proton-pyrophosphatase as determined by radiation inactivation. Biochem. J. 316, 143-147.
24. Veldhoven, P. P. V., and Mannaert G. P. (1987) Inorganic and organic phosphate measurement in the nanomolar range. Analytical Biochemistry. 161, 45-48.
25. Walker, R. R., and Leigh, R. A. (1981) Mg2+-dependent, action stimulated inorganic pyrophosphatase associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.). Planta 153, 150-155.
26. Walmsley, A. R. (2000) Fluorescence spectroscopy for monitoring changes in the conformation of membrane transporter. Membrane Transport Baldwin, S. A., ed. Oxford University Press, New York, pp. 167-169.
27. Wang, M. Y., Leight, R. A., Kaestner, K. H. and Sze, H. (1986) Electrogenic H+-pumping pyrophosphatase in tonoplast vesicles of oat roots. Plant Physiol. 81, 497-502.
28. Ward, A., Sanderson, N. M., Rutherford, N. G., Poolman, B., and Henderson, P. J. F. (2000) The amplified expression, identification, purification, assay, and properties of hexahistidine-tagged bacterial membrane transport proteins. Membrane Transport Baldwin, S. A., ed. Oxford University Press, New York, pp. 141-166.
29. White, P. J., Marshall, J., and Smith, J. A. C. (1990) Substrate kinetics of the tonoplast H+-translocating inorganic pyrophosphatase and its activation by free Mg2+. Plant Physiol. 93, 1063-1070.
30. Yang, S. J., Jiang, S. S., Tzeng, C. M., Kuo, S. Y., Hung, S. H., and Pan, R. L. (1996) Involvement of tyrosine residue in the inhibition of plant vacuolar H+-pyrophosphatase by tetranitromethane. Biochim. Biophys. Acta 1294, 89-97.
31. Yang, S. J., Jiang, S. S., Van, R. C., Hsiao, Y. Y., and Pan, R. L. (2000) A lysine residue involved in the inhibition of vacuolar H+-pyrophosphatase by fluorescein 5'-isothiocyanate. Biochim. Biophys. Acta 1460, 375-83.
32. Zhen, R. G., Kim, E. J., and Rea, P. A. (1997) Acidic residues necessary for pyrophosphate-energized pumping and inhibition of the vacuolar H+-pyrophosphatase by N,N'-dicyclohexylcarbodiimide. J. Biol. Chem. 272, 22340-22348.

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