Abstract
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.