臺灣博碩士論文加值系統

已知植物液泡膜上焦磷酸水解酶(簡稱V-PPase)在維持細胞質的中性酸鹼度扮演很重要的角色：V-PPase 藉由水解焦磷酸驅動質子由細胞質輸入液泡中，以維持細胞質的中性酸鹼度。由於對於焦磷酸水解酶的結構尚未清楚，因此根據TopPred Ⅱ軟體所預測的地誌模式，受質水解的催化位置被認為是位在第五與第六穿膜區之間的環區且面向在細胞質中。目前吾人還不清楚是否此催化區會與相鄰的穿膜區互相影響，或是與穿膜區上個別的胺基酸有相互關係。第六穿膜區在不同物種間有高度相似性，並且可能參與催化功能或維持結構的穩定性。在此篇論文,我們利用點突變的方法，分別將21個胺基酸置換成丙氨酸，在酵母菌內異體表現綠豆的焦磷酸水解酶，並測其活性與質子傳送作用以觀察第六穿膜區的重要性。結果發現Y299A, E301A,A306S, L307A, L317A 與N318A突變株不論水解活性或是偶合效率均嚴重受損。這些突變株在第六穿膜區的位置恰巧分布在兩端與中間，且其在整各螺旋環柱的分佈均分三區。由此猜測，這三區具有維持穩定結構的功能。A305S, V308A, A310S, G316A與H319A仍保有活性但降低了偶合效率，且位在螺旋環柱的同一面，暗示這些胺基酸可能與質子運送有關。另外，藉由離子效應的研究，我們也發現Y299A, C304A, L307A, V308A, L317A與N318A突變株對於鉀離子、鈣離子、鈉離子與氟離子均不敏感，暗示可能參與這些離子的結合。而E301突變株在離子效應除了氟離子以外也有同樣的情形。在酵素酶切實驗中，A305S, A306S, L307A, 與N318A突變株較能抵抗酵素的酶切反應;其中除了A305S 以外的突變株水解活性都很低。因此，我們認為這些突變株的低水解活性是由於其蛋白構型改變所導致。

The vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1) plays a significant role in the maintenance of pH value of cytoplasm via the proton translocation from cytosol to vacuolar lumen at the expense of PPi hydrolysis. The overall structure of the membranes-associated PPases is unclear. According to the topology of V-PPase prediced by TopPred Ⅱ, the motif of catalytic site for substrate hydrolysis was presumably located in loop V and exposed to the cytosol. It was not certain whether the motif of catalytic site interacts with adjacent transmembrane domains (TM), nor is the detailed correlation between the different amino acids. TM VI is relatively conserved and believed to participate in catalytic function and conformation stability. In this study, we carried out alanine scanning mutagenesis along TM VI of the mung bean V-PPase. Different amino acids were substituted singly by alanine and a series of site-direct mutants were constructed, over-expressed in Saccharomyces cerevisiae, and their enzymatic activities and proton translocations were determined. Y299A, E301A, A306S, L307A, L317A and N318A mutants exhibited gross impairment of both PPi hydrolysis and coupling efficiencies. The deterioration of these reactions at both ends and the middle of TM Ⅵ and the location of these amino acids at trisection of the helix wheel suggest their roles in maintaining the stability of conformation. A305S, V308A, A310S, G316A, and H319A exhibited an injury of proton translocation ability and show a decrease in the coupling efficiency but not PPi hydrolysis. They also locate at one face of the helix wheel, implicating their involvement in proton translocation. Studies on ion effects, it showed that the Y299A, C304A, L307A, V308A, L317A and N318A mutants are less sensitive to the K+, Ca2+, Na+, and F-, suggesting they exert effects on binding of these ions. Further evidence indicates that E301 may be involved in binding of these ions but not F-. From the proteolysis analysis, the A305S, A306S, L307A, and N318A mutants were relatively resistant the proteolysis. Moreover, their PPi hydrolysis activities were significantly inhibited expect A305S mutant. We suggest the low enzymatic activity of these mutants is due to the conformational change.

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Materials and Methods-------------------------------------6
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References-----------------------------------------------29
Figures and Tables---------------------------------------33

Ausubel, F. M., Brent, R., Kingston, R., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1999) Short Protocols in Molecular Biology, Unit: 13.7 pp. 31-35. 4th Ed, John Wiley & Sons Inc., Canada.
Barik, S. (1997) PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering (White, B. A. eds) pp.173-182, Humana Press Inc., New Jersey, USA.
Baykov, A. A., Dubnova, E. B., Bakuleva, N. P., Evtushenko, O. A., Zhen, R. G., and Rea, P. A. (1993) Differential sensitivity of membrane-associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme. FEBS Lett. 327:199-202
Belogurov, G. A., and Lahti, R. (2002) A lysine substitute for K+. A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans. J. Biol. Chem. 277:49651-49654
Drozdowicz, Y. M., and Rea, P. A. (2001) Vacuolar H+ pyrophosphatases: from the evolutionary backwaters into the mainstream. Trends Plant Sci. 6:206-211
Hsiao, Y. Y., Van, R. C., Hung, H. H., and Pan, R. L. (2002) Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue. J. Protein Chem. 21:51-58
Hung, S. H., Chiu, S. J., Lin, L. Y., and Pan, R. L. (1995) Vacuolar H+-pyrophosphatase cDNA (Accession No. U31467) (PGR 95-082) from etiolated mung bean seedlings. Plant Physiol. 109:1-125
Kim, Y., Kim, E. J, and Rea, P. A. (1994) Isolation and characterization of cDNA encoding the vacuolar H+-pyrophosphatase of Beta vulgaris. Plant Physiol. 106:375-382
Kim, E. J, Zhen, R. G., and Rea, P. A. (1994) Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport. Proc. Natl. Acad. Sci. USA. 91:6128-6132
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
Kuo, S. Y., and Pan, R. L. (1990) An essential arginyl residue in the tonoplast pyrophosphatase from etiolated mung bean seedings. Plant Physiol. 93:1128-1133
Laemmli, U. K. (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature (London) 222:680-685
Larson, E., Howlett, B., and Jagendorf, A. T. (1986) Artificial reductant enhancement of the Lowry method for protein determination. Anal. Biochem. 155:243-248
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. 196:11-17
Maeshima, M. (2000) Vacuolar H+-pyrophosphatase. Biochim. Biophys. Acta 1465:37-51
Maeshima, M. (2001) Tonoplast transporters: organization and function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52:469-497
Maruyama, C., Tanaka, Y., Takeyasu, K., Yoshida, M., and Sato, M. H. (1998) Structural studies of the vacuolar H+-pyrophosphatase: sequence analysis and identification of the residues modified by fluorescent cyclohexylcarbodiimide and maleimide. Plant Cell Physiol. 39:1045-1053
Nakanishi, Y., Saijo, T., Wada, Y., and Maeshima, M. (2001) Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase. J. Biol. Chem. 276:7654-7660
Nishi, T., and Forgac, M. (2002) The vacuolar (H+)-ATPases--nature's most versatile proton pumps. Nat. Rev. Mol. Cell Biol. 3:94-103
Rea, P. A., Britten, C. J., Jennings, I. R., Calvert, C. M., Skiera, L. A., Leigh, R.A., and Sanders, D. (1992) Regulation of vacuolar H+-pyrophosphatase by free calcium. Plant Physiol. 100:1706-1715
Salisbury, F. B., and Ross, C.W. (1992) Plant Physiology, pp. 24-25.4th edition, Wadsworth Inc., California, USA.
Tzeng, C.M., Yang, C.Y., Yang, S.J., Jiang, S.S., Kuo, S.Y., Hung, S.S., Ma, J.T., Pan, R.L., (1996) Subunit structure of vacuolar proton-pyrophosphatase as determined by radiation inactivation, Biochem. J. 316:143–147
Walker, R. R., and Leigh, R. A. (1981) Mg2+-dependent, cation-stimulated inorganic pyrophosphatase associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.). Planta 153:150-155
Wu, J. J., Ma, J. T., and Pan, R. L. (1991) Functional size analysis of pyrophosphatase from Rhodospirillum rubrum determined by radiation inactivation. FEBS Lett. 283:57-60
Yang, S. J., Jiang, S. S., Tzeng, C. M., Kuo, S. Y., and Pan, R. L. (1996) Involvement of tyrosine residues in the inhibition of plant vacuolar H+-pyrophosphatase by tetranitromethane. Biochim Biophy. Acta 1294:89-97
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 1460:375-383
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