Proton pumping activity in intact vacuoles purified from grape protoplasts. The accumulation of protons was determined by measuring the fluorescence quenching of ACMA [14, 15] in 2 × 105 vacuoles. Typical fluorescence signals of the initial velocities of proton pumping by V-H+-PPase after addition of 0.1 to 150 μM PPi (A) and the corresponding Michaelis-Menten plot (B). Michaelis-Menten plot of the initial velocities of proton pumping by V-H+-ATPase as a function of ATP concentration (0.05 to 1.5 mM) (C). The results presented in B and C show the average values of two independent experiments.