Purification and charcacterization of plasma membrane inorganic H+-pyrophosphatase from mung bean (Vigna radiata L.) seedlings

• Main Authors: Tu, Shuh Long, 涂世隆
• Other Authors: Pan, Rong Long
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/77595878116686078320

碩士 === 國立清華大學 === 輻射生物研究所 === 84 === 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.