A Ca2+-ATPase and a Mg2+/H+-antiporter are present on tonoplast membranes from roots of Zea mays L.

Abstract

Abstract.The primary or secondary energized transport of Ca 2+, Mg 2 § and H § into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca 2+ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca 2+ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Cae+-pumping and tonoplast H+-pyrophosphatase (PP~ase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca2 § activity could be detected. The ATP-dependent Ca 2 § uptake into tonoplast vesicles (using Ca 2+ concentrations from 0.8-1 gM) was completely inhibited by the Ca 2 § ionophore ionomycin (1 gM) whereas the protonophore nigericin (1 gM) which eliminates ATPdependent intravesicular H § accumulation had no effect. Vanadate (IC5o = 43 gM) and diethylstilbesterol (IC50 =5.2 laM) were potent inhibitors of this type of Ca 2+ 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 Ca 2 § transport mechanism is the only functioning Ca 2 § transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca 2+ concentration. In contrast, the secondary energized Ca2+-transport mechanism of the tonoplast, the low-affinity Ca 2 § § which was reported to allow the uptake of Ca 2+ in exchange for H § functions chiefly as an Mg 2 § transporter under physiological conditions because cytosolic Mg 2+ is several orders of magnitude higher than the Ca 2+ concentration. This conclusion was deduced from experiments showing that Mg 2 § ions in a concentration range of 0.01 to 1 mM Abbreviations: DES=diethylstilbesterol; DCCD=N,N'-dicyclohexylcarbodiimide; CCR = cytochrome-c reductase; IC5o = concentration which causes 50% inhibition; IDPase=inosine diphosphatase; Indo 1-1-(2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2 '-amino-5' methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid; PP~ase = tonoplast H +-pyrophosphatase Correspondence to: A. Hager; FAX: 49(7071) 293287 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 Ca 2 § differently from and independently of the Mg 2+ concentration. Calcium was a strong inhibitor for the H § (IC50 = 18 ~tM, Hill coefficient nH=l.7) but a weak one for the H+-ATPase (IC50 = 330 gM, nH= 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca 2 § and Mg2+-regulated H § PPiase, (ii) the newly described high-affinity CaZ+-ATPase, (iii) the low-affinity Mg2+(Ca2+)/H+-antiporter and (iv) the H §

A calmodulin‐stimulated Ca2+‐ATPase from plant vacuolar membranes with a putative regulatory domain at its N‐terminus1

“…BCAI represents the first plant Ca 2 + -ATPase gene cloned so far for which biochemical evidence exists for the Ca2+ -pumping nature of its gene product. The presence of Ca 2 + -ATPases in the vacuolar membrane has been reported previously [3,23,24]. This would imply the presence of at least two Ca2+ -transport systems in this membrane: one is the BCAI Ca2+ -ATPase, and the other is a Ca 2 +/H+ antiporter [25].…”

Modulation of an Intracellular Calmodulin-Stimulated Ca2+-Pumping ATPase in Cauliflower by Trypsin (The Use of Calcium Green-5N to Measure Ca2+ Transport in Membrane Vesicles)

“…A Ca2+/nH+ antiporter is responsible for CaZt transport into the vacuole, whereas Ca2+-ATPases are located in the plasma membrane and the ER (Evans et al, 1991;Chanson, 1993;Askerlund and Sommarin, 1996). Ca2+-ATPases may also be present in the vacuolar membrane and the inner plastid envelope (Gavin et al., 1993;Huang et al, 1993;Pfeiffer and Hager, 1993). In animals, only the plasma-membrane Ca2+-ATPase is directly stimulated by CaM (Carafoli, 1994).…”

Electrochemical Potential Gradients of H+, K+, Ca2+, and Cl- across the Tonoplast of the Green Alga Eremosphaera Viridis

“…Pfeiffer and Hager, 1993). There is increasing evidence that Ca2+ release from the vacuole is involved in Ca2+ signaling (Schumaker and Sze, 1987a;Evans et al, 1991;Bush, 19931, and an inositol 1,4,5-trisphosphate-activated Ca2+ channel was discovered in the tonoplast (Alexandre et al, 1990).…”