Plasma Membrane Ca-ATPase of Radish Seedlings

Abstract

In this work, we exploited the capability of the plasma membrane Ca-ATPase to utilize ITP as a substrate to study its characteristics in plasma membrane vesicles purified from radish (Raphanus sativus L.) seedlings. The Demonstrating the hydrolytic activity of the PM Ca-ATPase in native PM vesicles has proven difficult, due to the simultaneous operation of the much more active PM H+-ATPase, which is inhibited by Ca2", already in the micromolar range (5). Taking advantage of the different pH optima of the PM H+-ATPase (approximately 6.5) and of the PM CaATPase (7.0-7.5), we have shown that a Ca2e-dependent ATPase activity inhibited by submicromolar EB is localized at the PM ofradish seeds (21). The biochemical characteristics ofthis activity so far investigated, as well as its functional mol wt determined by radiation-inactivation, are very similar to those determined for ATP-dependent Ca2`uptake into PM vesicles, indicating that it represents the hydrolytic activity of the PM Ca-ATPase (21-23). However, the simultaneous operation of the Ca2t-inhibited PM H+-ATPase is an important source of error in these measurements because any treatment can be expected to influence both activities in the same or in the opposite sense. Because the PM H+-ATPase is highly specific for ATP as a substrate (5), it should be possible to use ITP or GTP as a substrate to measure the hydrolytic activity of the PM Ca-ATPase in native PM vesicles, minimizing complications arising from the simultaneous operation of the H+-ATPase. Analysis of GTP-dependent Ca2" uptake into PM vesicles from red beet has shown that GTP is a useful means to probe the transport function of the PM Ca-ATPase (27), but no data are available on its suitability to measure the hydrolytic activity of this enzyme. Because the presence of regulatory GTP-binding

Cytosolic Concentration of Ca2+ Regulates the Plasma Membrane H+-ATPase in Guard Cells of Fava Bean.

“…However, the latter possibility is unlikely because the activity of Ca2+-ATPase in the membranes was blocked by 1 pM erythrosin B, which had been included in the reaction mixture, as shown in Figure 4. Hydrolysis activity of ITP, a specific substrate for Ca2+-ATPase (Carnelli et al, 1992), increased with the increase of Ca2+ concentration, which is indicative of plasma membrane Ca2+-ATPase (Carnelli et al, 1992), and this Ca2+-dependent activity was inhibited almost completely by erythrosin B at 1 pM ( Figure 4). ITP hydrolysis was inhibited by vanadate, and inhibition was greater than that by erythrosin B, probably because vanadate inhibits both Ca2+-and H+-ATPases and because a portion of ITP hydrolysis is catalyzed by H+-ATPase (Briskin, 1990;Carnelli et al, 1992;Cocucci and Marre, 1984).…”

“…Ca2+-ATPase activity was measured by determining the Pi released from ITP, a specific substrate for Ca2+-ATPase, according to the method of Carnelli et al (1992) with modifications. The reaction mixture (100 pL) contained 10 mM Mops-KOH, pH 7.0, 0.25 M mannitol, 5 mM MgCIz, 1 mM EGTA, 50 mM KN03, 5 pglmL oligomycin, 0.5 mM ammonium molybdate, membrane preparation (20 pg of which was protein), free Caz++, and the indicated concentrations of erythrosin B (Figure 4).…”

Plasma Membrane Ca-ATPase of Radish Seedlings

“…Methods for seed germination, PM purification, and protein determination are given in the accompanying paper (9).…”

“…Measurements of Ca2 Uptake ATP-and ITP-dependent Ca2`uptake were assayed as described in the accompanying paper (9 Measurements of Ca2+-Dependent ITPase Activity Assays were performed in the presence of 75 jig mL-' Brij 58 essentially as described in the accompanying paper (9) in assay media buffered at pH 6.9 or 7.5 with 40 mM BTPHepes. Free Ca2`concentrations in media containing different CaCl2 concentrations and 1 mm (at pH 6.9) or 2 mm (at pH 7.5) EGTA were computed using the values of apparent association constant of the Ca-EGTA complex (5 x 105 M-' at pH 6.9 and 7.6 x 106 M-' at pH 7.5) determined in the accompanying paper (9).…”

Effect of preparations Methyure and Ivine on Са(2+)-ATPases activity in plasma and vacuolar membrane of corn seedling roots under salt stress conditions

“…2+ -АТPase was determined spectrophotometrically (SF2000, λ = 600 nm) by the amount of released Р i [19], using special substrate inositoltriphosphate (ITP), activity was expressed in nmol Р i /mg protein/min. Transport activity in the plasma and vacuolar membrane was measured by fluorescent method [20] (sound Fluo 4 AM) [21], using spectrofluorometer Quanta Master 40 PTI (Canada) under excitation of 495 nm, emis sion 522 nm with the software FelixGX 4.1.0.3096 presenting in ∆%F/mg protein/min.…”