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研究生(外文):Wei-Zhen Lee
論文名稱(外文):Characterization of Transmembrane Domain 11 of the Mung Bean Vacuolar H+-Pyrophosphatase by Alanine-substituted Mutagenesis
指導教授(外文):Rong-Long Pan
外文關鍵詞:vacuolepyrophosphatasetransmembrane domainhelical wheelsite-directed mutagenesis
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植物及真菌類細胞存在著大型的液泡,液泡膜上帶有兩種氫離子唧筒,分別是液泡焦磷酸水解酶 (簡稱V-PPase) 及液泡腺苷三磷酸水解酶 (簡稱V-ATPase)。它們負責將氫離子由細胞質送入液泡中,產生膜電位差。V-PPase以簡單、低成本的焦磷酸 (簡稱PPi) 為受質,轉成輸送氫離子所需的能量。此外在結構上,V-PPase亦是一約80 kDa、且具14-16穿膜區段之多肽鏈組成。這樣的酵素無異提供一個很棒的模型,幫助我們研究「水解PPi」與「輸送氫離子」之間的偶合機制。在這篇論文中,我們使用「丙胺酸替換突變法」,將綠豆V-PPase在第十一穿膜區內之所有胺基酸逐一替換。所產生的突變株被送入酵母菌內進行異體表現,並從中萃取微粒體,此微粒體內含大量突變的V-PPase,可供我們進行各項試驗。首先,我們一一測其「PPi水解活性」及「氫離子傳送作用」,並以此算出偶合效率。然而在這些突變株中,唯有K541A的酵素活性有明顯下降,此氨基酸 (Lys-541) 對V-PPase的重要性是可想而知。 此外,我們也找出五個胺基酸,分別是Ser-547、Leu-553、Leu-555、Phe-556及Ala-558,可能與酵素的偶合機制有關。接下來,我們繼續對這些突變株的其他特性進行探討。在「離子效應」的實驗中,鉀離子可促進酵素的活性,但促進的倍數隨各個突變株而異。在「熱穩定性」的實驗中,我發現V560A 及S561A這兩個突變株對熱的抵抗力比野生株來的高。另外,在酵素酶切實驗中,野生株與所有突變株皆可被酵素切動,然而,一旦加入V-PPase之受質,此受質將保護野生株與所有突變株的V-PPase不被酵素所切。收集這些實驗數據,可幫助我們對V-PPase的作用機制有更多的了解。
A vacuole is a membrane-enclosed fluid-filled cavity found in the cells of plants and fungi. There are two proton pumps in the tonoplast, vaculoar H+-pyrophosphatases (V-PPase; EC and vaculoar H+-ATPase (V-ATPase; EC They play a main role in catalyzing electrogenic H+-translocation from the cytosol to the vacuolar lumen to generate an inside-acidic and inside-positive membrane potential. V-PPase uses a simple, low-cost substrate pyrophosphate (PPi) as an energy source. Meanwhile, V-PPase contains only a single type of polypeptide of 80 kDa with 14-16 transmembrane domains (TMs) rendering a good model for studying the coupling mechanism between proton translocation and PPi hydrolysis. Among these transmembrane domains, TM 11 is highly conserved in many organisms. In this study, the residues in TM 11 of mung bean V-PPase were mutated by means of alanine-substituted mutagenesis. Mutated genes were over-expressed in Saccharomyces cerevisiae, and the V-PPase-enriched microsomes were prepared. The PPi hydrolysis activities, proton translocation, and coupling efficiency of the mutant V-PPases were then determined. K541A was the only mutant whose enzymatic activity decreased dramatically. It is apparent that the residue Lys-541 is important for the function of V-PPase. In addition, five significant residues (Ser-547, Leu-553, Leu-555, Phe-556 and Ala-558) were identified presumably to be involved in coupling mechanism. Furthermore, the ion effects, thermostability, and proteolytic analysis of these mutants were measured. There are some variations in degree of K+-stimulation for these mutants. Besides, we also found that the thermal stability of V560A and S561A is much higher than wild type. The susceptibilities of several mutant V-PPases to trypsin digestion in the absence of physiological substrate Mg2PPi were different, suggesting the variation in their conformation. The importance of each amino acid residues along TM11 is discussed. A working model of V-PPase is proposed accordingly.
Materials and Methods---------7
Figures and Tables-----------33
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