質子傳送-焦磷酸水解酶是一種質子傳送幫浦，運用了簡
單的無機小分子—焦磷酸做為能量的來源，來驅動質子通過
細胞膜。從破傷風桿菌中發現的質子傳送-焦磷酸水解酶擁有
十六個跨膜結構，而且在整個蛋白質中只發現了三個色氨酸。
色氨酸是一種擁有芳香族環的氨基酸，同時對許多蛋白質來
說是很重要的氨基酸。因此，我們藉由色氨酸定點取代突變
法來研究色氨酸在破傷風桿菌中質子傳送-焦磷酸水解酶的
角色。首先，我們先測定經由色氨酸定點取代突變法所突變
的質子傳送-焦磷酸水解酶中的焦磷酸水解活性、傳送質子的
能力、及質子偶合效率。用組氨酸取代在75 位置上的色氨
酸後，發現了質子傳送能力大幅地下降，這表示說，蛋白質
在結構上有明顯的改變。再者，我們測量了一些常見的離子
(K+、Ca2+、Na+、F-)在突變後質子傳送-焦磷酸水解酶中的影
響。添加鈣離子、鈉離子、氟離子及抽離鉀離子，皆會造成
野生株及突變株某種程度上質子水解活性的下降。破傷風桿
菌中質子傳送-焦磷酸水解酶的功能及反應機制在本研究中
能獲得相應的闡明。

H+-translocating inorganic pyrophosphatase (H+-PPase), a
member of proton pumps, utilizes inorganic pyrophosphate (PPi) as energy source to generate proton-motive forces across membranes. H+-PPase from Clostridium tetani (CtH+-PPase) contains 16 transmembrane domains and shows only three tryptophan residues in entire protein. Tryptophan is an important aromatic amino acid for many proteins. Therefore, we investigated the role of tryptophan residues in CtH+-PPase by means of tryptophan-substitution mutagenesis. The PPi hydrolysis activities, proton translocations, and coupling efficiencies of the mutated CtH+-PPases were then determined. Substitution for W75 showed a dramatic decrease in coupling ratio, indicating obvious structural changes of the protein. Furthermore, the effect of common ions (K+, Ca2+, Na+, F-) added to these mutated CtH+-PPases were measured. Addition of Ca2+, Na+, F- and subtraction of K+ would diminish the PPi hydrolysis activities in wild type and mutants to a certain extent.
The function and conformational change of CtH+-PPase were
accordingly elucidated in this study.

References
Baltscheffsky, M., Nadanaciva, S., and Schultz, A. (1998) A
pyrophosphate synthase gene: molecular cloning and sequencing of the cDNA encoding the inorganic pyrophosphate synthase from Rhodospirillum rubrum. Biochim. Biophys. Acta 1364:301-306
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
Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248- 254
Carden, D. E., Walker, D. J., Flowers, T. J., and Miller, A. J. (2003) Single-cell measurements of the contributions of cytosolic Na+ and K+ to salt tolerance. Plant Physiol. 131:676-683 19
Clerc S., and Barenholz Y. (1998) A quantitative model for using acridine orange as a transmembrane pH gradient probe. Anal. Biochem. 259:104-111
Drozdowicz, Y. M., Lu, Y. P., Patel, V., Fitz-Gibbon, S., Miller, J. H., and Rea, P. A. (1999) A thermostable vacuolar-type membrane pyrophosphatase from the archaeon Pyrobaculum aerophilum implications for the origins of pyrophosphate-energized pumps. FEBS Lett. 460:505-512
Fiske C. H., and Subbarow Y. (1925) The colorimetric determination of phosphorus. J. Biol. Chem. 66:375- 400
Gordon-Weeks, R., Steele, S. H., and Leigh, R. A. (1996) The role of magnesium, pyrophosphate, and their complexes as substrates and activators of the vacuolar H+-pumping inorganic pyrophosphatase. Plant Physiol. 111:195-202 20
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
Hsiao Y. Y., Van R. C., Hung S. H., Lin H. H., and Pan R. L. (2004) Roles of histidine residues in plant vacuolar H+-pyrophosphatase. Biochim. Biophys. Acta 1608:190-199
Ikeda M, Rahman M. H, Moritani C, Umami K, Tanimura Y,
Akagi R, Tanaka Y, Maeshima M, and Watanabe, Y. (1999) A
vacuolar H+-pyrophosphatase in Acetabularia acetabulum: Molecular cloning and comparison with higher plants and a bacterium. J. Exp. Bot. 50:139-140
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 21
Kirsch, R. D. and Joly, E. (1998) An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes. Nucleic Acids Res. 26:1848-1850
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
Maeshima, M. (2000) Vacuolar H+-pyrophosphatase. Biochim. Biophys. Acta 1465:37-51
Rea P. A., and Poole R. J., (1985) Proton-translocating inorganic pyrophosphatase in red beet (Beta vulgaris L.) tonoplast vesicles. Plant Physiol. 77:46-52
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 22
Rottenberg H. (1979) The measurement of membrane potential anddelta pH in cells, organelles, and vesicles, in: S. Fleischer, L. Packer (Eds.), Methods Enzymol., vol. 55, Academic Press, New York, pp. 547-569
Sarafian, V., and Poole, R. J. (1989) Purification of an
H+-translocating inorganic pyrophosphatase from vacuole membranes of red beet. Plant Physiol. 91:34-38
Wang, B., Luttge, U., and Ratajczak, R. (2001) Effects of salt treatment and osmotic stress on V-ATPase and V-PPase in leaves of the halophyte Suaeda salsa. J. Exp. Bot. 52:2355-2365
Wang M. Y., Lin Y. H., Chow W. M., Chung T. P., and Pan R. L. (1989) Purification and characterization of tonoplast ATPase from etiolated mung bean seedlings. Plant Physiol. 90:475-481 23
Yang, S. J., Jiang, S. S., Tzeng, C. M., Kuo, S. Y., 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
Yazaki Y., Asukagawa N., Ishikawa Y., Ohta E., and Sakata M.
(1988) Estimation of cytoplasmic free Mg2+ levels and phosphorylation potentials in mung bean root tips by in vivo 31P NMR spectroscopy. Plant Cell Physiol. 29:919-924
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