Abstract
The H +-translocating inorganic pyrophosphatase (H +-PPase) of Beta vacuolar membrane (tonoplast) vesicles has been purified by 90-fold from detergent-solubilized membranes and the MgPP i-binding subunit identified by affinity labeling. The purified enzyme has a specific activity of 1100 μmol mg·h and contains one prevalent M i = 64000 polypeptide which strictly copurifies with activity. Treatment of tonoplast vesicles with N-ethylmaleimide (NEM) inhibits the PPase with pseudo-first-order kinetics. Inclusion of MgPP i in the reaction medium diminishes the pseudo-first-order rate constant ( k o) by more than 30-fold while free PP i increases k o by about 2-fold. Pretreatment of tonoplast vesicles with [ 12C]NEM in the presence of MgPP i followed by incubation with [ 14C]NEM in the presence of MgPP ior free PP i yields a single polypeptide of M i = 64000 which shows MgPP i-protectable, PP i-potentiated 14C-labeling and comigration with PPase activity during gel filtration. It is deduced that the M r = 64000 polypeptide constitutes the MgPP ibinding subunit of the H +-PPase. Purification; Substrate-binding subunit; Tonoplast; Vacuolar membrane; Pyrophosphatase, H +-translocating inorganic
Published Version
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