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研究生:蕭義勇
研究生(外文):Yi-Yuong Hsaio
論文名稱:保留性組胺酸和精胺酸在植物液泡質子輸送焦磷酸水解酵素之功能角色探討
論文名稱(外文):Roles of Conserved Histidine and Arginine Residues in Plant Vacuolar H+-Pyrophosphatase
指導教授:潘榮隆潘榮隆引用關係
指導教授(外文):Rong-Long Pan
學位類別:博士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:102
中文關鍵詞:液泡質子輸送焦磷酸水解酵素組胺酸精胺酸化學修飾抑制基因定點突變
外文關鍵詞:Vacuolar H+ -pyrophosphataseHistidinediethylpyrocarbonateArgininephenylglyoxal23-Butanedione
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中文摘要
植物液泡(Vacuole)在植物細胞中主要功能為細胞質恆定之維持及儲存生理代謝物質。植物液泡執行其功能時主要藉由液泡膜系上各種通道或輸送性蛋白來完成,其中有兩種磷酸水解酵素:液泡H+-ATP水解酵素(vacuolar H+-ATPase)和液泡H+-PPase水解酵素(Vacuolar H+-PPase)為驅動這些液泡之通道或輸送性蛋白的能量轉換裝置,因為其能量來源主要由ATP和PPi提供。目前植物液泡H+-ATP水解酵素(H+-ATPase)之研究在其功能和生理角色上已經有相當豐富的成果和了解。但液泡H+-PPase水解酵素(H+-PPase)至目前仍有很大研究了解的空間。本研究就從化學修飾抑制及基因定點突變的角度,來了解液泡H+-PPase水解酵素之化學修飾抑制作用之成因,藉以標定酵素中功能性胺基酸的位置和角色。並可提供研究膜蛋白質或酵素之結構與功能之概念和方法。
首先以組胺酸(Histidine)之專一化學修飾劑diethylpyrocarbonate
(DEPC)對綠豆液泡H+-PPase進行化學修飾抑制研究,顯示DEPC可以抑制綠豆液泡H+-PPase之PPi水解酵素活性及質子輸送功能。且綠豆液泡H+-PPase之受質Mg2+-PPi 可保護綠豆液泡H+-PPase之酵素活性中心不被DEPC之化學修飾抑制,顯示可能有DEPC修飾之組胺酸為綠豆液泡H+-PPase之功能上重要胺基酸基團。另外,已知綠豆液泡Cys-630為酵素活性中心鄰近的胺基酸基團,已被證實Cys-630可被其專一性化學修飾劑NEM修飾但不會抑制酵素活性,利用此修飾劑進行先期化學修飾再利用DEPC對綠豆液泡H+-PPase進行抑制動力學研究,顯示DEPC對綠豆液泡H+-PPase抑制值由1.1histidine/subunit下降至0.7 histidine /subunit,顯然NEM修飾作用可以減緩DEPC對綠豆液泡H+-PPase抑制作用。綜合上述結果,我們認為可能有一個DEPC修飾之組胺酸鄰近或位於酵素活性中心。
基於以上結果,我們要進一步確認DEPC修飾之標的組胺酸位置,而利用基因定點突變,和酵母菌異體基因表現與微小體(Microsome)分離,且從酵母菌基因體序列中並未比對出可能為H+-PPase存在酵母菌基因體中,經西方點墨(Western bolt),PPi水解活性鑑定及質子輸送之測定結果顯示液泡H+-PPase是可以大量被遞送(Targeting)到酵母菌內膜系統中且具有液泡H+-PPase之活性,故我們基於這些基本條件進行異體基因表現研究。經由胺基酸序列比對發現有6個組胺酸存在綠豆液泡H+-PPase中,其中H716在不同生物之H+-PPase胺基酸序列中為唯一具有高度保留性之組胺酸,經基因定點突變和酵母菌異體基因表現,當H716被置換成H716A之定點突變株會降低PPi水解活性和質子輸送功能。經由計算其PPi水解/質子輸送效率顯示比正常株下降約50%,再者H716A突變也會改變液泡H+-PPase之PPi最佳水解環境之pH值,有0.8單位的酸性偏移現象,顯示Histidine被置換成Alanine後液泡H+-PPase之PPi水解活性和質子輸送功能會受影響。已知F-可藉由形成Mg2+-F-2複合物會干擾液泡H+-PPase之PPi水解活性,實驗結果顯示H716A突變會降低F-抑制PPi水解活性作用達正常株之1/2。由熱處理T1/2值之評估得知各種Histidine置換成Alanine之突變株並未看出有明顯改變H+-PPase之熱穩定性,H716A亦然。如將H716A之突變株再用DEPC進行化學修飾抑制,顯示DEPC抑制性只有相對於正常株之1/3,而其他突變株未顯示相當之耐受性,亦即DEPC修飾主要對象是H716。另外,利用Trypsin 進行部分水解(limited digestion)研究顯示Mg2+-PPi未能在H716A突變株上呈現與正常株一樣的受質保護現象,顯示H716A有可能引發酵素活性中心結構改變以致使酵素受質保護現象消失,但也不排除因結構改變而間接影響受質保護現象。以上結果顯示H716可能參與液泡H+-PPase之PPi水解活性及質子輸送功能或是酵素活性之調節功能上,且H716也是DEPC修飾抑制液泡H+-PPase活性主要成因。
其次,前人研究結果顯示利用精胺酸(Arginine)專一性化學修飾劑phenylglyoxal(PGO)及2,3-Butanedione(BD)進行化學修飾,能抑制液泡H+-PPase之PPi水解活性及質子輸送功能,由抑制動力學研究顯示可能有一個或一個以上之化學修飾之精胺酸為液泡H+-PPase之功能上必要之基團,此基團亦會因Mg2+-PPi保護酵素活性中心而降低化學修飾抑制作用。因此,引發我們有興趣要鑑定出此化學修飾之標的,首先由胺基酸序列比對發現共有15個精胺酸存在綠豆液泡H+-PPase中,再進行基因定點突變及酵母菌異體基因表現和微小體分離獲得各種精胺酸定點突變之液泡H+-PPase進行分析。目前已標的出R242很可能為此化學修飾之標的,因為R242A突變使液泡H+-PPase之PPi水解活性及質子輸送功能喪失95%之活性,在最佳PPi水解環境之pH值之觀察,R242A突變會引發最佳PPi水解環境pH值有1.0單位之酸性偏移的現象,進一步觀察精胺酸專一性化學修飾劑PGO及BD之修飾抑制顯然對R242A突變株並未產生抑制作用,在高濃度之F-存在下觀察PPi水解活性作用,F-抑制R242A突變株只有正常之株之1/6,綜合上述結果,我們相信R242應該是精胺酸化學修飾劑抑制之主要對象,但也不能排除因失去電價性側鏈引發結構改變之間接影響。總之,我們結合化學修飾抑制及基因定點突變成功的標的出功能性胺基酸之位置及其角色如何,也提供蛋白質結構功能研究上,另一種研究方法及概念。
Abstract

Vacuolar H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at expense of PPi hydrolysis. The identification of gene encoding an amino acid sequence demonstrates that vacuolar H+-PPase of mung bean contains 6 histidine residues and 15 arginine residues. This study showed vacuolar H+-PPase may contain several histidine and arginine residue(s) essential for the enzymatic activity and H+-translocation of vacuolar H+-PPase. Furthermore, we identified the roles of histidine and arginine residues in mung bean vacuolar H+-PPase by site-directed mutagenesis. A line of mutants with histidine and arginine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among histidine mutants scrutinized, only did the mutation of H716 decrease the enzymatic activity, the proton transport and the coupling ratio of vacuolar H+-PPase. The mutation at H716 of vacuolar H+-PPase shifted the optimum pH value but not the T1/2 (pretreatment temperature at which half enzymatic activity is observed) for PPi hydrolytic activity. Mutation of H716 is obviously declined the effect of substrate protection on the vacuolar H+-PPase as determined by immunoblotting analysis after limited trypsin digestion. Moreover, mutation of these histidine residues modified the inhibitory effects of F- and Na+, but not that of Ca2+. Single substitution of H704, H716 and H758 by alanine released the effect of K+ stimulation, indicating possible location of K+ binding in the vicinity of domains surrounding these residues. A working topological model is thus proposed to elucidate the roles of crucial histidines.
As for arginine mutated variants, R242A, R523A, and R609A mutants displayed declined activity of PPi hydrolysis and proton translocation than the wild-type. These mutants showed a shift in optimal pH for enzymatic activity. Among arginine mutants, R242A is relatively resistant to PGO and BD treatments, suggesting that it is the primary target for the attack of these modifiers. Furthermore, only did R242A mutant displayed relatively lower sensitivity to F- inhibition under similar conditions. Taken together, we speculate that R242 may locate in active domain of vacuolar H+-PPase. A working model is proposed to accommodate information from previous studies on the catalytic site of vacuolar H+-PPase.
目錄 (Contents)
List of Tables..........................................iv
List of Figures.........................................v
List of Appendix........................................viii
誌 謝..................................................ix
Abbreviations........................................... 1
中文摘要................................................ 3
Abstract................................................ 6
I. Introduction......................................... 8
II. Materials and methods...............................15
1. Preparation of vacuolar H+-PPase from mung bean seedlings...............................................15
2. Modification and labeling stoichiometry of vacuolar
H+-PPase by DEPC.......................................16
3. Site-directed mutagenesis............................17
4. Microorganisms for site-directed mutagenesis.........18
5. Preparation of vacuolar H+-PPase-enriched microsomes from yeast cells........................................19
6. Enzyme assay and protein determination...............20
7. Measurement of proton translocation..................21
8. SDS/PAGE and Western analysis........................22
9. Trypsin proteolysis..................................22
10. Chemicals...........................................23
III. Results.................................................24
Section one: Role of essential histidine in H+-PPases..................................................24
1. Chemical modification of vacuolar H+-PPase...........24
1.1. Inactivation of H+-PPase by DEPC...................24
1.2. Protection against DEPC inhibition.................25
1.3. Stoichiometry of DEPC labeling.....................26
2. Site-directed mutagenesis studies....................27
2.1. Heterologus expression and characterization of H+-PPases..................................................27
2.2. Expression and H+-PPase activities for histidine mutants.................................................29
2.3. Kinetic properties of mutants......................30
2.4. Sensitivities of mutants to DEPC...................31
2.5. Ion effects on enzymatic activities of histidine mutants.................................................32
2.6. Thermal stability and proteolytic analysis of histidine mutants.......................................33
Section two: Role of essential Arginine in H+-PPases....34
1. Expression and H+-PPase activities for arginine residues................................................34
2. Kinetic properties of mutant.........................36
3. Sensitivities of mutants to phenylglyoxal and 2,3 butanedione.............................................37
4. Ion effects on enzymatic activities..................38
5. Thermal stability of mutants........................39
IV. Discussion..........................................40
1. Roles of histidine residues in vacuolar H+-PPase.....40
2. Roles of conserved arginine residues in vacuolar H+-PPase...................................................46
3. A working model for active domain of vacuolar H+-PPase...................................................50
V. References...........................................52
Tables..................................................61
Figures.................................................68
Appendix................................................98
V. References

Baltscheffsky, M., Schultz, A., and Baltscheffsky, H. H+-PPases: a tightly membrane-bound family, FEBS Lett. 457 (1999) 527-533.

Barik, S. Site-directed mutagenesis by double polymerase chain reaction: megaprimer method, in: B. A. White (Ed.) Methods of Molecular Biology, vol.15, Humana Press, New Jersey, 1993, pp.277-286.

Baykov, A. A., Dubnova, E. B., Bakuleva, N. P., Evtushenko, O. A., Zhen, R -G., and Rea, P. A. Differential sensitivity of membrane-associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme, FEBS Lett. 327 (1993) 199-202.

Belogurov, G.A., and Lahti, R. A lysine substitute for K+: A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans, J. Biol. Chem. 277 (2002) 49651-49654.

Bienengraeber, M., Echtay, K.S., and Klingenberg, M. H+-transport by uncoupling protein (UCP-1) is dependent on a histidine pair, absent in UCP-2 and UCP-3, Biochemistry 37 (1998) 3-8.

Bragg, P. D., and Hou, C. The role of conserved histidine residues in the pyridine nucleotide transhydrogenase of Escherichia coli, Eur. J. Biochem. 241 (1996) 611-618.

Brandsch, M., Brandsch, C., Ganapathy, M. E., Chew, C. S., Ganapathy, V., and Leibach, F.H. Influence of proton and essential histidyl residues on the transport kinetics of the H+/peptide cotransport systems in intestine (PEPT 1) and kidney (PEPT 2), Biochim. Biophys. Acta 1324 (1997) 251-262.

Buckley, J. T., Wilmsen, C., Lesieur, A., Schulze, F., Pattus, Parker, M. W., and van der Goot, F. G. Protonation of histidine-132 promotes oligomerization of the channel-forming toxin aerolysin, Biochemistry 34 (1995) 16450-16455.

Chanchevalap, S., Yang, Z., Cui, N., Qu, Z., Zhu, G., Liu, C., Giwa, L. R., Abdulkadir, L., and Jiang, C. Involvement of histidine residues in proton sensing of ROMK1 channel, J. Biol. Chem. 275 (2000) 7811-7817.

Claros, M.G., and Heijne, G. von TopPred II: an improved software for membrane protein structure predictions, Comput. Appl. Biosci. 10 (1994) 685-686.

Cooperman, B. S., Baykov, A. A., and Lathi, R. Evolutionary conservation of the active site of the soluble inogganic pyrophosphatase, Trands Biochem. Sci. 17 (1992) 262-266.

Drozdowicz, Y. M., Lu, Y. P., Patel, V., Fitz-Gibbon, S., Miller, J. H., and Rea, P. A. A thermostable vacuolar-type membrane pyrophosphatase from the archaeon Pyrobaculum aerophilum: implications for the origins of pyrophosphate-energized pumps, FEBS Lett. 460 (1999) 505-512.

Drozdowicz, Y. M., Kissinger, J. C., and Rea, P. A. AVP2, a sequence-divergent, K+-insensitive H+-translocating inorganic pyrophosphatase from Arabidopsis, Plant Physiol. 123 (2000) 353-362.

Drozdowicz, Y. M., and Rea, P. A. Vacuolar H+- pyrophosphatases: from the evolutionary backwaters into the mainstream, Trends Plant Sci. 6 (2001) 206-211.

Fiske, C. H., and Subbarow, Y. The colorimetric determination of phosphorous, J. Biol. Chem. 66 (1925) 378-400.

Gietz, R. D., Schiestl, R. H., Willems, A. R., and Woods, R. A. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure, Yeast 11 (1995) 355-360.

Gordon-Weeks, R., Steele, S. H., and Leigh, R. A. The role of magnesium, pyrophosphate, and their complexes as substrates and activators of the vacuolar H+-pumping inorganic pyrophosphatase, (Studies using ligand protection from covalent inhibitors), Plant Physiol. 111 (1996) 195-202.

Hsiao, Y. Y., Van, R. C., Hung, S. H., Lin, H. H., and Pan, R. L. Roles of histidine residues in plant vacuolar H+-pyrophosphatase, Biochim. Biophys. Acta 1608 (2004)190-199.

Hsiao, Y. Y., Van, R. C., Hung, H. H., and Pan, R. L. Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue, J. Protein Chem. 21 (2002) 51-58.

Hoffman, K., and Stoffel, W. TMbase – A database of membrane spanning proteins segments, Biol. Chem. Hoppe-Seyler 347 (1993) 166 (abstr.).

Jones, D. T., Taylor, W. R., and Thornton, J. M. A model recognition approach to the prediction of all-helical membrane protein structure and topology, Biochemistry 33 (1994) 3038-3049.

Kasher, J. S., Allen, K. E., Kasamo, K., and Slayman, C. W. Characterization of an essential arginine residue in the plasma membrane H+-ATPase of Neurospora crassa, J. Biol. Chem. 261 (1986) 10808-10813.

Kim, E. J., Zhen, R -G., and Rea, P. A. Site-directed mutagenesis of vacuolar H+-pyrophosphatase: necessity of Cys634 for inhibition by maleimides but not catalysis, J. Biol. Chem. 270 (1995) 2630-2635.

Kim, E. J., Zhen, R -G., and Rea, P. A. Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport, Proc. Natl. Acad. Sci. U.S.A. (1994) 6128-6132.

Kuo, S. Y., and Pan, R. L. An essential arginyl residue in the tonoplst pyrophosphatase from etiolated mung bean seedlings, Plant Physiol. 93 (1990) 1128-1133.

Laemmli, U. K. Cleavage of structure proteins during the assembly of the head of bacteriophage T4, Nature (London) 222 (1970) 680-685.

Larson, E., Howlett, B., and Jagendorf, A. T. Artificial reductant enhancement of the Lowry method for protein determination, Anal. Biochem. 155 (1986) 243-248.

Maeshima, M. TONOPLAST TRANSPORTERS: Organization and Function Ann. Rev. Plant Physiol. Plant Mol. Biol. 52 (2001) 469-497.

Maeshima, M. Vacuolar H+-pyrophosphatase, Biochim. Biophys. Acta 1465 (2000) 37-51.

Maeshima, M. H+-translocating inorganic pyrophosphatase of plant vacuoles: inhibition by Ca2+, stabilization by Mg2+ and immunological comparison with other inorganic pyrophosphatases, Eur. J. Biochem. 196 (1991) 11-17.

Maruyama, C., Tanaka, Y., Mitsuda, N. T., Takeyasu, K., Yoshida M., and Sato, M. H. Structural studies of the vacuolar H+-pyrophosphatase: sequence analysis and identification of the residues modified by fluorescent cyclohexylcarbodiimide and maleimide, Plant Cell Physiol. 39 (1998)1045–1053.

Matile, P. Biochemistry and function of vacuoles, Ann. Rev. Plant Physiol. 29 (1978)193-213.

Mattinoia, E. Transport processes in vacuolar of higher plants, Bot. Acta 105 (1992) 232-245.

Martinoia, E., Massonneau, A., and Frangne, N. Transport processes of solutes across the vacuolar membrane of higher plants, Plant Cell Physio. 41 (2000)1175-86.

Miles, E.W. Modification of histidyl residues in proteins by diethylpyrocarbonate, Meth. Enzymol. 47 (1977) 431-442.

Nakanishi, Y., Saijo, T., Wada, Y., and Maeshima, M. Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase, J. Biol. Chem. 276 (2001) 7654-7660.

Okada, A., Miura, T., and Takeuchi, H. Protonation of histidine and histidine-tryptophan interaction in the activation of the M2 ion channel from influenza A virus, Biochemistry 40 (2001) 6053-6060.

Patterson, A. R., Wada, T., and Vik, S. B. His15 of subunit a of the Escherichia coli ATP synthase is important for the structure or assembly of the membrane sector Fo, Arch. Biochem. Biophys. 368 (1999) 193-197.

Rajan, S., Wischmeyer, E., Liu, X. G., Preisig-Müller, R., Daut, J., Karschin, A., and Derst, C. TASK-3, a novel tandem pore domain acid-sensitive K+ channel: an extracellular histidine as pH sensor, J. Biol. Chem. 275 (2000) 16650-16657.

Rea, P. A., Kim, Y., Sarafian, V., Poole, R. J., Davies, J. M., and Sanders, D. Vacuolar H+ translocating pyrophosphatases: a new category of ion translocase, Trends Biochem. Sci. 17 (1992) 348-353.

Rea, P. A., and Poole, R. J. Vacuolar H+-tranclocating pyrophosphatase, Ann. Rev. Plant Physiol. Plant Mol. Biol. 44 (1993) 157-180.

Rea, P. A., and Poole, R. J. Chromatographic resolution of H+-translocating pyrophosphatase from H+-translocation ATPase of higher plant tonoplast. Plant Physiol. 81 (1986) 126-129.

Rea, P. A., and Sanders, D., Tonoplast energization: Two H+ pumps, one membrane, Physiol. Plantarum 71 (1987) 131-141.

Reinhold, L., and Kaplan, A. Membrane transport of sugars and amino acids, Ann. Rev. Plant Physiol. 35 (1984) 45-83.

Sams, C. F., and Matthews, K. S. Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding, Biochemistry 27 (1988) 2277-2281.

Sarsfina, V., and Poole, R. J. Purification of an H+-translocating inorganic pyrophosphatase from vacuole membranes of red beet, Plant Physol. 91 (1989) 34-38.

Schmidt L. A., and Briskin, D. P. Energy transduction in tonoplast vesicles from red beet (Beta vulgaris L.) storage tissue: H+/Substrate stochiometries for the H+-ATPase and H+-PPase, Arch. Biochem. Biophys, 301(1993)165-17

Schultz, A., and Baltscheffsky, M. Properties of mutated Rhodospirillum rubrum H+-pyrophosphatase expressed in Escherichia coli, Biochim. Biophys. Acta 1607 (2003) 141-151.

Sez, H. H+-translocating ATPase: advances using membranevesicles, Ann. Rev. Plant Physiol, 36 (1985)175-208.

Takeshiga, K., and Hager, A. Ion effects on the H+-translocating adenosine triphosphatase and pyrophosphatase associated with the tonoplast of Chara coralline, Plant Cell Physiol. 29 (1988) 649-657.

Thompson, J. D., Higgins, D. G., and Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res. 22 (1994) 4673-4680.

Tzeng, C. M., Yang, C. Y., Yang, S. J., Jiang, S. S., Kuo, S. Y., Hung, S. S., Ma, J. T., and Pan, R. L. Subunit structure of vacuolar proton- pyrophosphatase as determined by radiation inactivation, Biochem. J. 316 (1996) 143-147.

Walker, R. R., and Leigh, R. A. Mg2+-dependent, cation-stimulated inorganic pyrophosphatase associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.), Planta 153 (1981) 150-155.

Wang, M. Y., Lin, Y. H., Chow, W. M., Chung, T. P., and Pan, R. L. Purification and characterization of tonoplast H+-ATPase from etiolated mung bean seedlings, Plant Physiol. 90 (1989) 475-481.

Wanger, G. J., and Mulredy, P. Characterization and solubilization of nucleotide-specific, Mg2+-ATPase and Mg2+-pyrophosphatase of tonoplast, Biochem. Biophys. Acta 728(1983)267-280.

Wikström, M. Mechanism of proton translocation by cytochrome c oxidase: a new four-stroke histidine cycle, Biochim. Biophys. Acta 1458 (2000) 188-198.

Wink, M. The plant vacuolar: a multifunctional compartment, J. Exp. Bot. 44 (1993) 231-246.

Yang, S. J., Jiang, S. S., Van, R. C., Hsiao, Y. Y., and Pan, R. L. A lysine residue involved in the inhibition of vacuolar H+-pyrophosphatase by fluorescein 5'-isothiocyanate, Biochim. Biophys. Acta 1460 (2000) 375-383.

Yang, S. J., Jiang, S. S., Kuo, S. Y., Hung, S. H., Tam, M. F., and Pan, R. L. Localization of a carboxylic residue possibly involved in the inhibition of vacuolar H+-pyrophosphatase by N, N'-dicyclohexylcarbodi-imide, Biochem. J. 342 (1999) 641-646.

Yang, S. S., Kuo, S. S., Tsai, Y. R., Yang, S. J., Jiang, S. S., Kuo, S. Y., Hung, S. H., and Pan, R. L. Subunit interaction of vacuolar H+-pyrophosphatase as determined by high hydrostatic pressure, Biochem. J. 331 (1998) 395-402.

Yang, S. J., Jiang, S. S., Tzeng, C. M., Kuo, S. Y., Hung, S. H., and Pan, R. L. Involvement of tyrosine residue in the inhibition of plant vacuolar H+-pyrophosphatase by tetranitromethane, Biochim. Biophys. Acta 1294 (1996) 89-97.

Zhang, Y., Pines, G., and Kanner, B. I. Histidine 326 is critical for the function of GLT-1, a (Na+/ K+)-coupled glutamate transporter from rat brain, J. Biol. Chem. 269 (1994) 19573-19577.

Zhen, R -G., Kim, E. J., and Rea, P. A. Acidic residues necessary for pyrophosphate-energized pumping and inhibition of the vacuolar H+-pyrophosphatase by N,N'-dicyclohexylcarbodi-imide, J. Biol. Chem. 272 (1997) 22340-22348.

Zhen, R -G., Kim, E. J., and Rea, P. A. Localization of cytosolically oriented maleimide-reactive domain of vacuolar H+-pyrophosphatase, J. Biol. Chem. 269 (1994) 23342-23350.
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1. 3. 王政彥,2001,社區終身學習合作網路的發展,成人教育,第62期,pp.11~21
2. 5. 李青蓉,2003/6,探討教育中高齡者使用電腦軟體技能的方法雨教材設計,管理與資訊學報,第八期,pp.165~193
3. 7. 李青蓉,2002,初探中高齡學習者學習電腦技能之較學方式與過程﹘以上機課混合網路教學為例,教學科技與媒體,pp.38~51
4. 8. 李青松,2002,高齡者休閒參與之研究,中華家政學刊,第31期,pp.21~38
5. 11. 江雪齡,1994,發揮空中教育的效果,成人教育,第19期,pp.40~43
6. 16. 林奇賢,1997,全球資訊網輔助學習系統﹘網際網路與國小教育,資訊與教育雜誌,第58期,pp.2~11
7. 17. 林振春,1999,學習型社區中的高齡者學習方案,成人教育,第49期,pp.15~22
8. 18. 林彥呈,許家斌,王宗興,管倖生,張育銘,陳國祥,鄧怡莘,2000/11,網頁要素對性認知影響之研究,工業設計,第28卷,第二期,pp.122~128
9. 24. 林麗惠,2003,高齡者參與高等教育知教育之決定模式,成人教育,pp.13~21
10. 27. 洪榮昭,1994,銀髮族的能力開發,成人教育,第17期,pp.39~41
11. 29. 胡夢鯨,1994,台灣地區成人教育現代化進程中的困境與出路,成人教育,第19期,pp.34~39
12. 33. 陳清美,2001,高齡者的學習特性,成人教育,第63期,pp.45~49
13. 34. 陳清美,2001/11/20,高齡學對學習環境的偏好,成人教育,第64期,pp.43~51
14. 35. 陳清美,2003,高齡者學習組織的合齡與分齡初探,成人教育,第72期,pp.22~30
15. 37. 陳建志,1999,為高齡者設計的因子與評估方法之探討,明志技術學院學報,第31卷,pp.83~91