A Ca-ATPase and a Mg/H-antiporter are present on tonoplast membranes from roots of Zea mays L.

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