A CA2+-ATPASE AND A MG2+/H+-ANTIPORTER ARE PRESENT ON TONOPLAST MEMBRANES FROM ROOTS OF ZEA-MAYS L

The primary or secondary energized transport of Ca2+, Mg2+ and H+ into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca2+ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca2+ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Ca2+-pumping and tonoplast H+-pyrophosphatase (PP(i)ase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca2+-pumping activity could be detected. The ATP-dependent Ca2+ uptake into tonoplast vesicles (using Ca2+ concentrations from 0.8-1 muM) was completely inhibited by the Ca2+ ionophore ionomycin (1 muM) whereas the protonophore nigericin (1 muM) which eliminates ATP-dependent intravesicular H+ accumulation had no effect. Vanadate (IC50 = 43 muM) and diethylstilbesterol (IC50 = 5.2 muM) were potent inhibitors of this type of Ca2+ transport. The nucleotides GTP, UTP, ITP, and ADP gave 27%-50% of the ATP-dependent activity (K(m) = 0.41 mM). From these results, it was suggested that this ATP-dependent high-affinity Ca2+ transport mechanism is the only functioning Ca2+ transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca2+ concentration. In contrast, the secondary energized Ca2+-transport mechanism of the tonoplast, the low-affinity Ca2+/H+-antiporter, which was reported to allow the uptake of Ca2+ in exchange for H+, functions chiefly as an Mg2+ transporter under physiological conditions because cytosolic Mg2+ is several orders of magnitude higher than the Ca2+ concentration. This conclusion was deduced from experiments showing that Mg2+ ions in a concentration range of 0.01 to 1 mM triggered a fast efflux of H+ from acid-loaded vesicles. Furthermore, the proton-pumping activity of the tonoplast H+-ATPase and H+-PP(i)ase was found to be influenced by Ca2+ differently from and independently of the Mg2+ concentration. Calcium was a strong inhibitor for the H+-PP(i)ase (IC50 = 18 muM, Hill coefficient nH = 1.7) but a weak one for the H+-ATPase (IC50 = 330 muM, nH = 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca2+- and Mg2+-regulated H+-PP(i)ase, (ii) the newly described high-affinity Ca2+-AT-Pase, (iii) the low-affinity Mg2+(Ca2+)/H+-antiporter and (iv) the H+-ATPase.