臺灣博碩士論文加值系統

植物液泡是一種多功能的胞器，在成熟的高等植物中，其含量約佔細胞總體積的80~90%以上。當外在環境改變時，為維持細胞質內部生理的恆定，由液泡所形成的內在環境會有某些程度的調整；一般而言，液泡的主要功能在於維持細胞的膨壓、酸鹼平衡以及代謝物的儲存等。
植物液泡囊膜上包含兩種傳送質子的幫浦：液泡質子腺核三磷酸水解酵素(V-ATPase)與液泡無機焦磷酸水解酵素(V-PPase)。液泡質子腺核三磷酸水解酵素廣泛存在於真核細胞中，而液泡無機焦磷酸水解酵素則只能在植物及一些細菌中找到，其主要功能是利用水解PPi所產生的能量將質子從液泡膜外傳送至膜內，形成pH梯度與膜電位差，藉由這些力量可以驅動離子及代謝物進出液泡囊膜。
在本實驗中，我們萃取白化綠豆下胚軸中的液泡，先分別利用兩種detergent (sodium deoxycholate, lysophosphatidylcholine)將V-PPase從液泡膜上溶離出來，再藉由gel filtration與ion exchange 層析法作進一步純化。以其純化所得的液泡無機焦磷酸水解酵素為材料，用carboxypeptidase Y (CPY)處理，導致酵素C端的末端缺失，來探討C端在整個酵素功能與結構之間所扮演的角色。
由實驗結果得知，相較於未純化的液泡囊膜無機焦磷酸水解酵素，經過CPY處理之後，純化的蛋白質活性受到明顯的抑制(65%)。純化的酵素之所以會對CPY那麼的敏感，主要歸因於缺乏液泡囊膜的保護。同時，質子傳送能力也受到嚴重的破壞(50%)，而呈現decoupling的現象。
另外，經CPY處理之純化的液泡囊膜無機焦磷酸水解酵素，並不再受到鉀離子的激活，因此，我們進而推測無機焦磷酸水解酵素的C端可能含有鉀離子的結合或調控位置。而且，無機焦磷酸水解酵素的生理受質，焦磷酸鹽，對於C端的缺失反應具有保護作用。
綜合以上的結果，我們可以發現無機焦磷酸水解酵素的C端可能與陽離子的結合或調控有關，同時，扮演著維持酵素質子通道結構完整的角色。位於活性中心的受質結合部位，與酵素C端產生某種交互作用，而形成堅韌的構形以避免CPY的攻擊。

Vacuoles are multifunctional organelles in the mature plant cells and occupy 80~90% of total cell volume. They are important in the homeostasis of the plant cells, which involves the regulation of cell turgor, cytoplasmic pH, and storage of metabolites.
Plant vacuolar vesicles contain two parallel proton pumps, namely vacuolar H(+)-pumping adenosine-triphosphatase (V-ATPase; EC 3.6.1.3), which is widely distributed in most eukaryotes, and vacuolar H(+)-pumping inorganic pyrophosphatase (V-PPase; EC 3.6.1.1), which exists primarily in higher plants and several bacteria. V-PPase catalyzes PPi hydrolysis and the electrogenic H(+)-translocation from the cytosol to the vacuolar lumen to generate an inside-acidic pH gradient (pH) and an interior-positive membrane potential () for the transport of ions and secondary metabolites.
Vacuolar H(+)-PPase was purified from etiolated hypocotyls of mung bean seedlings (Vigna radiata L.) by solubilization with sodium deoxycholate and lysophosphatidylcholine, gel filtration as well as ion exchange chromatography.
In this study, the treatment of carboxypeptidase Y (CPY) markedly suppressed H+ translocation and purified vacuolar H(+)-PPase activity instead of membrane-bound enzymatic activity in a concentration-dependent manner. It was indicated that the purified vacuolar H(+)-PPase is more sensitive to CPY owing to the removal of the membrane. In addition, CPY obviously decoupled the association of proton translocation and enzymatic activity, suggesting that the C-terminus of the enzyme either participates directly in proton translocation or plays a role in the maintenance of the structure in the proton channel.
Furthermore, the incubation with CPY relieved the stimulation of KCl, implying that the C-terminus is most likely the candidate for the region involved in the K(+)-binding and/or probably the K(+)-regulation of the enzyme. On the other hand, the protection studies revealed that PPi is crucial for most protection of vacuolar H(+)-PPase against C-terminal digestion by CPY.
As a result, we speculate that the C-terminus of vacuolar H(+)-PPase is not directly involved in the catalytic activity, but provides the site for the cation-binding and regulation as well as supports the structure of a proton channel. Moreover, PPi binding site in catalytic domain may exert long-distance interaction with C-terminus of vacuolar H(+)-PPase, resulting in a tougher conformation against proteolytic attack by CPY.

Page
1. Abbreviations 1
2. Abstract 3
3. Introduction 8
4. Materials and Methods
4-1. Plant materials 17
4-2. Tonoplast preparation 17
4-3. Purification of vacuolar H+-pyrophosphatase 19
4-4. Protein determination and enzyme activity assay 20
4-5. PPi-dependent proton pumping assay 21
4-6. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis 22
4-7. Immunoblot assay 23
4-8. Cleavage of vacuolar H+-PPase by carboxypeptidase Y 24
4-9. Chemicals 25
5. Results
5-1. Purification of the vacuolar H+-PPase 26
5-2. Immunoblotting with the anti-pyrophosphatase antibody 26
5-3. Inhibition of PPi hydrolysis by carboxypeptidase
Y 27
5-4. Inactivation of proton translocation by
carboxypeptidase Y 28
5-5. Effects of K+ on vacuolar H+-PPase altered by
carboxypeptidase Y 28
5-6. Protection effect against carboxypeptidase Y 29
6. Discussion 31
7. References 35
8. Figures and Tables 44

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