H(+)-translocating inorganic pyrophosphatase of plant vacuoles. Inhibition by Ca2+, stabilization by Mg2+ and immunological comparison with other inorganic pyrophosphatases.
The effects of divalent cations, especially Ca2+ and Mg2+, on the proton-translocating inorganic pyrophosphatase purified from mung bean vacuoles were investigated to compare the enzyme with other pyrophosphatases. The pyrophosphatase was irreversibly inactivated by incubation in the absence of Mg2+. The removal of Mg2+ from the enzyme increased susceptibility to proteolysis by trypsin. Vacuolar pyrophosphatase required free Mg2+ as an essential cofactor (K0.5 = 42 microM). Binding of Mg2+ stabilizes and activates the enzyme. The formation of MgPPi is also an important role of magnesium ion. Apparent Km of the enzyme for MgPPi was about 130 microM. CaCl2 decreased the enzyme activity to less than 60% at 40 microM, and the inhibition was reversed by EGTA. Pyrophosphatase activity was measured under different conditions of Mg2+ and Ca2+ concentrations at pH 7. 2. The rate of inhibition depended on the concentration of CaPPi, and the approximate Ki for CaPPi was 17 microM. A high concentration of free Ca2+ did not inhibit the enzyme at a low concentration of CaPPi. It appears that for Ca2+, at least, the inhibitory form is the Ca2(+)-PPi complex. Cd2+, Co2+ and Cu2+ also inhibited the enzyme. The antibody against the vacuolar pyrophosphatase did not react with rat liver mitochondrial or yeast cytosolic pyrophosphatases. Also, the antibody to the yeast enzyme did not react with the vacuolar enzyme. Thus, the catalytic properties of the vacuolar pyrophosphatase, such as Mg2+ requirement and sensitivity to Ca2+, are common to the other pyrophosphatases, but the vacuolar enzyme differs from them in subunit mass and immunoreactivity.[1]References
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