Molecular Analysis and Characterization of H+-translocating Pyrophosphatase from Different Organisms
| Autor: | 張甫任 |
|---|---|
| Rok vydání: | 2013 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 101 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. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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