碩士 / 國立清華大學 / 生物資訊與結構生物研究所 / 92 / Vacuolar proton inorganic pyrophosphatase (V-PPase ; EC 3.6.1.1) is a membrane-bound protein, found primarily in higher plants and several bacteria. V-PPase can use the outlay of PPi hydrolysis to generate pH gradient across the tonoplast membrane to maintain the vacuoles in acidic condition. The His-tagged fusion protein makes it possible to purify the membrane-bound protein from the membrane by using the DDM (n-Dodecyl β-D-maltoside) as the detergent. With the application of affinity column Ni2+-NTA and FPLC system to purify the V-PPase, analysis by SDS-PAGE and Western blot reveal that the molecular weight of V-PPase is about 73 kDa.
In this studies, we compared the enzymatic activities of wild type membrane-bound V-PPase (mbWTVPP), His-tagged membrane-bound V-PPase (mbHisVPP), and purified His-tagged V-PPase (sHisVPP). The KM of mbWTVPP, mbHisVPP, and sHisVPP were 71.85 μM, 115.04 μM, and 250μM, respectively. The Vmax of these three types of V-PPase are 38.02 μmol PPi mg-1 h-1, 72.46 μmol PPi mg-1 h-1, 307.73 μmol PPi mg-1 h-1, respectively. The maximum enzymatic activity was measurement with the substrate of Mg2+/PPi ratio of 1:1 at pH 8.0. Furthermore, Na+, Ca2+, and F- inhibited the enzyme activities of these three types of V-PPase to the same level. These data showed that the purified His-tagged V-PPase was similar to the membrane-bound V-PPase. However, enzymatic activity of mbWTVPP was stimulated by about 10-fold; however, those of the mbHisVPP and sHisVPP were by about 2-3 fold. Obviously, the His-tagged fusion protein of V-PPase could modify the effect of K+ stimulation. Moreover, spectral studies indicate Mg2+ and Ca2+ could lead to the conformational change of the V-PPase. In further studies, we will use the Circular Dichroism (CD) to observe the secondary structure of V-PPase.
| Identifer | oai:union.ndltd.org:TW/092NTHU5112005 |
| Date | January 2004 |
| Creators | Shen Hsing Hsu, 徐慎行 |
| Contributors | Rong Long Pan, 潘榮隆 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 46 |
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