Abstract
ABSTRACT Plant vacuolar vesicles contain two parallel proton pumps; one is an enzyme common to all eucaryotes, vacuolar H+-ATPase (EC 3.6.1.3). Another is a novel H+-translocating pyrophosphatase (EC 3.6.1.1), which catalyzes both the hydrolysis of PPi and the electrogenic translocation of H+ from the cytosol to the lumen of the vacuole. The H+-PPase was successfully purified by two-step detergent solubilization and chromatography from etiolated hypocotyls of mung bean (Vigna radiata L.). The purified H+-PPase was at least 10-fold purification from the membrane vesicles. In this study, carboxypeptidase A (CPA) was used to investigate the relationship between structure and function of H+-PPase. Incubation of membrane H+-PPase with CPA caused a slight decrease in the rate of PPi hydrolysis with a 7-kDa segment from C-terminus digested as determined by Western blot analysis. However, purified vacuolar H+-PPase activity was obviously sensitive to CPA with a 50 % decreasing. H+-translocation was also markedly inhibited by 30 % in a concentration-dependent manner, probably due to the uncoupling of enzymatic reaction from proton pumping. The treatment of CPA abolished the stimulation of KCl but enhanced the inhibition of Ca2+, indicating that the C-terminus contains a K+-binding domain and probably exerts a long distant effect on Ca2+ regulation of the enzyme. An antibody specific to peptides corresponding to the putative substrate-binding site (DVGADLVGKVE) in the hydrophilic loop was prepared to investigate the functional domains of H+-PPase. Furthermore, a topological model is proposed to interpret essential roles of C-terminus of vacuolar H+-PPase. We thought that the C-terminal region of vacuolar H+-PPase is not directly involved in the catalytic activity, but maintains the structure of a proton channel as well as provides sites for the cation- binding and/or regulation.