A Ca2+-ATPase and a Mg2+/H+-antiporter are present on tonoplast membranes from roots ofZea maysL.

摘要

The primary or secondary energized transport of Ca2+, Mg2+and H+into tonoplast membrane vesicles from roots ofZea maysL. 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 (PPiase) 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 μM) was completely inhibited by the Ca2+ionophore ionomycin (1 μM) whereas the protonophore nigericin (1 μM) which eliminates ATP-dependent intravesicular H+accumulation had no effect. Vanadate (IC50= 43 μM) and diethylstilbesterol (IC50= 5.2 μM) were potent inhibitors of this type of Ca2+transport. The nucleotides GTP, UTP, ITP, and ADP gave 27–50 of the ATP-dependent activity (Km= 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+-PPiase was found to be influenced by Ca2+differently from and independently of the Mg2+concentration. Calcium was a strong inhibitor for the H+-PPiase (IC50= 18 μM, Hill coefficient nH = 1.7) but a weak one for the H+-ATPase (IC50= 330 μM, 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+-PPiase, (ii) the newly described high-affinity Ca2+-AT-Pase, (iii) the low-affinity Mg2+(Ca2+)/H+-antiporter and (iv) the