Study of the involvement of H+-dependent Ca2 uptake in intact vacuoles purified from grape protoplasts. Effect of Ca2+ on the pre-formed transmembrane proton gradient (A). Inset: Lineweaver-burk plot of the initial velocities of fluorescence recovery as function of Ca2+ concentration. The fluorescence signals shown are representative of at least three independent experiments. Intact vacuoles labelled with the calcium fluorescent probe Fluo-4 AM in the absence and in the presence of 300 μM calcymicin, observed under the fluorescence microscope (B).