Microsomal vesicles from 24-hour-old radish (Raphanas sativus L.) seedlings accumulate Ca2+ upon addition of MgATP. MgATP-dependent Ca"2 uptake co-migrates with the plasma membrane H'-ATPase on a sucrose gradient. Ca2" uptake is insensitive to oligomycin, inhibited by vanadate (ICs. 40 micromolar) and erythrosin B (ICo 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca2+ uptake is insensitive to protonophores. These results indicate that Ca2 transport in these microsomal vesicles is catalyzed by a Mg8-dependent ATPase localized on the plasma membrane. Ca2o strongly reduces ApH generation by the plasma membrane H'-ATPase and increases MgATP-dependent membrane potential difference (A4') generation. These effects of Ca2" on ApH and A4, generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca2" uptake into plasma membrane vesicles. The Ca2"-induced increase of A4, is collapsed by permeant anions, which do not affect Ca2"-induced decrease of ApH generation by the plasma membrane H'-ATPase. The rate of decay of MgATP-dependent ApH, upon inhibition of the plasma membrane H'-ATPase, is accelerated by MgATPdependent Ca2" uptake, indicating that the decrease of ApH generation induced by Ca2 reflects the efflux of H' coupled to Ca2" uptake into plasma membrane vesicles. It is therefore proposed that Ca2" transport at the plasma membrane is mediated by a Mg2"-dependent ATPase which catalyzes a nH'/Ca2" exchange.activated Ca2'-transport ATPase, whose proposed localization at the plasma membrane has not been supported with convincing experimental evidence (8,9,30). Secondary active systems, nH+/ Ca2`antiporters which utilize the AAH+ built up by a H+-pumping ATPase to drive Ca2+ transport (1,3,10,16,(20)(21)(22)(23)(27)(28)(29)(30), have been described in tonoplast vesicles isolated from higher plants and yeast (1,3,16,20,21,27) and in plasma membrane vesicles isolated from Neurospora (28).The starting point of this work was to ascertain whether the plasma membrane of higher plant cells is endowed with a nH+/ Ca2+ antiporter analogous to that described in Neurospora. We approached this problem using microsomal vesicles isolated from radish seedlings germinated for 24 h. We have previously demonstrated that microsomes isolated at this stage of development exhibit MgATP-dependent H+-pumping catalyzed only by the vanadate-sensitive plasma membrane H+-ATPase with no activity of the tonoplast H+-ATPase detectable (25). Thus, any contribution ofthe nH+/Ca2+ antiporter ofthe tonoplast to MgATPdependent Ca2' uptake in this membrane fraction is minimal.Consequently, this membrane fraction is well suited for investigating whether ApH generated by the plasma membrane H+-ATPase of higher plants drives active Ca2+ transport through a nH+/Ca2' antiporter.The data reported in this paper lead us to conclude that a nH+/Ca2+ antiport driven by ApH does not occur on the plasma membrane of radish seedlings. Our results indicate that Ca...

The transport and hydrolytic activities of the plasma membrane (PM) Ca 2 + pump were characterized in a PM fraction purified from seedlings of Arabidopsis thaliana by the aqueous two-phase partitioning technique. 10000-fold, EB 1000-fold and rose bengal only 50-fold more active on the Ca2+ pump than on the W -A'I'Pase): and iv) the effectiveness of EB in inhibiting the Ca 2 + pump was strongly affected by the protein concentration in the assay medium. KeywordsArabidopsis thaliana, plasma membrane, fluorescein derivatives, calmodulin, Ca 2 + pump. Ca2+ uptake could be energized by ATP and by lTP (at about 70 % the rate sustained by ATP). This characteristic was used to measure the hydrolytic activity of the enzyme as Ca 2 +-dependent ITPase activity. The PM Ca 2 + pump displayed a broad pH optimum around pH 7.2, was drastically inhibited by erythrosin B (EB), and was half-saturated by 60 pM ITP. It was stimulated by CaM, specially at low, non-saturating Ca 2 + concentrations. All of these characteristics closely resemble those of the PM Ca 2 + pump in other plant materials.Analysis of the effects of EB and other fluorescein derivatives (eosin Y and rose bengal) showed that: i) EB behaved as a competitive inhibitor with respect to ITP; ii) the PM Ca 2 + pump was drastically inhibited by concentrations of fluorescein derivatives (submicromolar), much lower than those required to inhibit the PM W -ATPase; iii) the different fluorescein derivatives were diversely efficient in inhibiting the activities of the Ca 2 + pump and of the W -ATPase of the PM (eosin Y was about Brij 58: BTP: CaM: EB: FCCP: HEPES: IC so : MES: PM: PMSF: Abbreviations and Symbols polyoxyethylene 20 cetyl ether bis-trispropane (1,3-bis(tris-(hydroxymethyl)methylamin0)-propane) calmodulin erythrosin B carbonyl cyanide p-trifluoromethoxyphenylhydrazone 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid effective concentration giving 50 % inhibition 4-morpholine-ethanesulfonic acid plasma membrane phenylmethyl-sulfonyl fluoride Bot. Acta 106 (1993) 20-25

ABSTRAC1The effect of fusicoccin on Mg:ATP-dependent H -pumping in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings was investigated by measuring the initial rate of decrease in the absorbance of the ApH probe acridine orange. Fusicoccin stimulated Mg:ATPdependent Hf-pumping when the pH of the assay medium was in the range 7.0 to 7.6 while no effect of fusicoccin was detected between pH 6.6 and pH 6.0. Both basal and fusicoccin-stimulated Hf-pumping were completely inhibited by vanadate and almost unaffected by nitrate. Fusicoccin did not change membrane permeability to protons and fusicoccininduced stimulation of Mg:ATP-dependent H -pumping was not affected by changes in the buffer capacity of the incubation medium. Deacetylfusicoccin stimulated Hf-pumping as much as fusicoccin, while the physiologically inactive derivative 8-oxo-9-epideacetylfusicoccin did not. Stimulation of Hf-pumping was saturated by 100 nanomolar fusicoccin. These data indicate that fusicoccin activates the plasma membrane H -ATPase by acting at the membrane level independently of the involvement of other cell components. The percent stimulation by fusicoccin was the same at all ATP concentrations tested (0.5-5.0 millimolar), thus suggesting that with fusicoccin there is an increase in V.. of the plasma membrane H'-ATPase rather than a decrease in its apparent K. for Mg:ATP.All of the physiological responses to FC2 appear to be related to a stimulation of electrogenic H+ extrusion mediated by the plasma membrane H+-ATPase. Stimulation ofthe plasma membrane H+-ATPase has been proposed as a primary step in the action of the toxin on the basis of evidence such as lack of a lag in the stimulating effect of FC on H+ extrusion and on hyperpolarization of AI, insensitivity of FC effects to inhibitors of RNA and protein synthesis, identification of specific FC-binding proteins at the plasma membrane and some stimulation by FC of the ATPase activity measured in microsomal fractions (for a review, see Refs. 17 and 18).In contrast to this view, it has been recently reported that FC induces in root hairs and in coleoptiles an early acidification of the cytoplasm, evident before any hyperpolarization of AI or stimulation of H+ extrusion is detectable (5, 7, 15). Since it has been shown that electrogenic H+ extrusion in vivo (6, 15, 28) well as the plasma membrane H+-ATPase in vitro (10,14,20,29) A more direct insight into the mechanism of action of FC can be provided by the study of its effects in vitro. This approach allows FC effects on membrane bound activities to be studied in the absence of other cell components. Recently, Rasi-Caldogno and Pugliarello (25), showed that FC strongly and reproducibly stimulated the H+-pumping and, to a lesser extent, the hydrolytic activity of the plasma membrane H+-ATPase in membrane vesicles isolated from radish seedlings. In this paper, we present a more detailed characterization of the stimulating effect of FC on the H+-pumping activity ofthe plasma membrane H+-ATPase ofradish. The ...

In microsomes from 24-hour-old radish (Raphanus sativus L.) seedlings ATP-dependent Ca2+ uptake occurs only in inside-out plasma membrane vesicles (F Rasi-Caldogno, MC Pugliarello, MlDe Michelis [1987] Plant Physiol 83: 994-1000). A Ca2 -dependent ATPase activity can be shown in the same microsomes, when assays are performed at pH 7.5. The Ca2 -dependent ATPase is stimulated by the Ca2+ ionophore A23187 and is localized at the plasma membrane. Ca2+-dependent ATPase activity and ATPdependent Ca2+ uptake present very similar saturation kinetics with erythrosin B (50% inhibition at about 0.1 micromolar), free Ca2+ (half-maximal rate at about 70 nanomolar), and MgATP (Km 15-20 micromolar). Ca2+ uptake can be sustained by GTP or ITP at about 60% the rate measured in the presence of ATP; only very low Ca2+ uptake is sustained by CTP or UTP and none by ADP. These results indicate that the Ca2+-ATPase described in this paper is the enzyme which drives active transport of Ca2+ at the plasma membrane of higher plants.

The Ca 2؉ -ATPase of the plasma membrane (PM) of germinating radish (Raphanus sativus L.) seeds was purified by calmodulin (CaM)-affinity chromatography using a batch procedure. PM purified by aqueous two-phase partitioning was solubilized with n-dodecyl ␤-D-maltoside and applied to a CaM-agarose matrix. After various washings with decreasing Ca 2؉ concentrations, the Ca 2؉ -ATPase was eluted with 5 mM ethylenediaminetetraacetate (EDTA). The EDTA-eluted fraction contained about 25% of the loaded Ca 2؉ -ATPase activity, with a specific activity 70-fold higher than that of the starting PM fraction. The EDTA-eluted fraction was highly enriched in a 133-kD polypeptide, which was identified as the PM Ca 2؉ -ATPase by 125 I-CaM overlay and fluoresceinisothiocyanate labeling. The PM Ca 2؉ -ATPase cross-reacted with an antiserum against a putative Ca 2؉ -ATPase of the Arabidopsis thaliana chloroplast envelope.