Abstract
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 couplesPPi hydrolysis with proton pumping, has been found on membranesof the tonoplast, chloroplast, mitochondria, and some speciesof bacteria. Here, we further demonstrated the existence of aH+-pumping PPase from mung bean plasma membrane. Enzymeactivity of PPase was observed on the plasma membrane andpossessed an optimum alkaline pH at 8.0-8.5. PPi-dependentproton translocation was concomitantly found on highly purifiedplasma membrane vesicles. A successful protocol includingplasma membrane preparation, detergent solubilization, gelfiltration, and anion exchange chromatographies was establishedto purify the enzyme. Analysis of size exclusion gel filtrationchromatography and SDS-PAGE revealed that plasma membrane H+-PPase was probably in a heterodimer form consisting of twosubunits of 65 and 67 kDa. The PPase activity was fluoride-sensitive, but could be stimulated by K+ and phospholipid usingMg2+ as a cofactor at Mg2+ / PPi ratio of 2 : 1. Takentogether, we believed that plasma membrane H+-PPase was a novelnew type of membrane-bound alkaline inorganic pyrophosphatase.