質子傳送-焦磷酸水解酶，是一種利用水解無機小分子—焦磷酸做為能量的來源，來驅動質子通過細胞膜的質子傳送幫浦。在這研究當中，我們建構出質子傳送-焦磷酸水解酶的表現載體，並利大腸桿菌為宿主去最佳化表現出三種細菌(Leptospira biflexa, Leptospira shermani, and Thermotoga maritima)中鑲嵌在膜上的焦磷酸水解酶，以利往後的分析實驗,像FRET、定點取代突變。首先，我們先測定不同株質子傳送-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。再者，我們測量了一些常見的離子(K+、Ca2+、Na+、F-)在不同株質子傳送-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，皆會造成三種焦磷酸水解酶某種程度上質子水解活性的下降。最後，我們收集和彙整每株焦磷酸水解酶的功能和特性，並建立一個對往後研究可輔助的系統。

H+-translocating inorganic pyrophosphatase (H+-PPase; EC 3.6.1.1) is a member of proton pumps that utilizes the hydrolysis of inorganic pyrophosphate (PPi) as energy source to pump protons across membranes. In this work, we have coloned H+-PPases into expression vector and tested the heterological expression of three integral membrane pyrophosphatases, all from bacterial sources, in Escherichia coli: Leptospira biflexa (LbH+-PPase), Leptospira shermani (LsH+-PPase), and Thermotoga maritima (TmH+-PPase), to obtain optimal expression level for further analysis, such as FRET、Site-directed mutagenesis. First, The PPi hydrolysis activities, proton translocations, and coupling efficiencies of the H+-PPases were determined from each H+-PPase. Furthermore, the effects of each common ions (K+, Ca2+, Na+, F-) added to these H+-PPases were measured, respectively. Addition of Ca2+, Na+, F- and deletion of K+ would diminish the PPi hydrolysis activities in LbH+-PPase, LsH+-PPase, and TmH+-PPase to a certain extent. Finally, we collected and organized the function and characterization of each H+-PPase to establish a workable system for further research of this proton pumping enzyme.

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