臺灣博碩士論文加值系統

位於膜上的無機焦磷酸水解酵素(pyrophosphatase)，目前已知其具有焦
磷酸水解及質子傳遞二種活性，而在液泡、葉綠體、粒線體的膜上，以及
一些細菌的細胞膜上，已經證實有無機焦磷酸水解酵素的存在。在本篇論
文中，我們嘗試從白化綠豆下胚軸的細胞膜上分離具有質子傳遞能力的無
機焦磷酸水解酵素。首先，我們證實了細胞膜上的確有無機焦磷酸水解酵
素的活性，而且在酸鹼值 8.0到 8.5之間達到最大。同時在高純度的細胞
膜囊泡上也發現了無機焦磷酸啟始的質子傳遞。因此我們接著進一步利用
清潔劑溶出無機焦磷酸水解酵素 (detergent solubilization)，再利用
分子篩 (gel filtration)及陰離子交換樹酯 (anion exchange)等管柱層
析法純化得到此酵素。由進一步的特性分析中，我們首先由可鑑定分子量
的分子篩管柱層析及SDS-PAGE中推測出此無機焦磷酸水解酵素是由二個65
及67 kDa的次單元所組成的異分子雙體 (heterodimer)。其活性必須存在
有鎂離子此種輔因子下才能維持，而且在鎂離子與無機焦磷酸是2比1的情
況下有最高活性。其活性受氟離子抑制；可以被鉀離子及磷脂刺激而提高
。由這些結果我們相信在細胞膜上的確存在有一種新的無機焦磷酸水解酵
素。

Membrane-bound inorganic pyrophosphatase (PPase), which couples
PPi hydrolysis with proton pumping, has been found on membranes
of the tonoplast, chloroplast, mitochondria, and some species
of bacteria. Here, we further demonstrated the existence of a
H+-pumping PPase from mung bean plasma membrane. Enzyme
activity of PPase was observed on the plasma membrane and
possessed an optimum alkaline pH at 8.0-8.5. PPi-dependent
proton translocation was concomitantly found on highly purified
plasma membrane vesicles. A successful protocol including
plasma membrane preparation, detergent solubilization, gel
filtration, and anion exchange chromatographies was established
to purify the enzyme. Analysis of size exclusion gel filtration
chromatography and SDS-PAGE revealed that plasma membrane H+-
PPase was probably in a heterodimer form consisting of two
subunits of 65 and 67 kDa. The PPase activity was fluoride-
sensitive, but could be stimulated by K+ and phospholipid using
Mg2+ as a cofactor at Mg2+ / PPi ratio of 2 : 1. Taken
together, we believed that plasma membrane H+-PPase was a novel
new type of membrane-bound alkaline inorganic pyrophosphatase.