Biomedical subjects

M C Pugliarello

Publications and source records attributed to M C Pugliarello.

18 recordsLinked to original sources

PubMed2000

Phenylarsine oxide inhibits the fusicoccin-induced activation of plasma membrane H(+)-ATPase.

To investigate the mechanism by which fusicoccin (FC) induces the activation of the plasma membrane (PM) H(+)-ATPase, we used phenylarsine oxide (PAO), a known inhibitor of protein tyrosine-phosphatases. PAO was supplied in vivo in the absence or presence of FC to radish (Raphanus sativus L.) seedlings and cultured Arabidopsis cells prior to PM extraction. Treatment with PAO alone caused a slight decrease of PM H(+)-ATPase activity and, in radish, a decrease of PM-associated 14-3-3 proteins. When supplied prior to FC, PAO drastically inhibited FC-induced activation of PM H(+)-ATPase, FC binding to the PM, and the FC-induced increase of the amount of 14-3-3 associated with the PM. On the contrary, PAO was completely ineffective on all of the above-mentioned parameters when supplied after FC. The H(+)-ATPase isolated from PAO-treated Arabidopsis cells maintained the ability to respond to FC if supplied with exogenous, nonphosphorylated 14-3-3 proteins. Altogether, these results are consistent with a model in which the dephosphorylated state of tyrosine residues of a protein(s), such as 14-3-3 protein, is required to permit FC-induced association between the 14-3-3 protein and the PM H(+)-ATPase.

Arsenicals↗

PubMed1989

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H-ATPase: I. Characteristics and Intracellular Localization of the Fusicoccin Receptor in Microsomes from Radish Seedlings.

The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a K(d) of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (K(d) 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H(+)-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H(+)-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H(+)-ATPase is mediated by fusicoccin binding to its plasma membrane receptor.

Journal Article↗

PubMed1989

Identification and Characterization of the Ca-ATPase which Drives Active Transport of Ca at the Plasma Membrane of Radish Seedlings.

In microsomes from 24-hour-old radish (Raphanus sativus L.) seedlings ATP-dependent Ca(2+) uptake occurs only in inside-out plasma membrane vesicles (F Rasi-Caldogno, MC Pugliarello, MI De Michelis [1987] Plant Physiol 83: 994-1000). A Ca(2+)-dependent ATPase activity can be shown in the same microsomes, when assays are performed at pH 7.5. The Ca(2+)-dependent ATPase is stimulated by the Ca(2+) ionophore A(23187) and is localized at the plasma membrane. Ca(2+)-dependent ATPase activity and ATP-dependent Ca(2+) uptake present very similar saturation kinetics with erythrosin B (50% inhibition at about 0.1 micromolar), free Ca(2+) (half-maximal rate at about 70 nanomolar), and MgATP (K(m) 15-20 micromolar). Ca(2+) uptake can be sustained by GTP or ITP at about 60% the rate measured in the presence of ATP; only very low Ca(2+) uptake is sustained by CTP or UTP and none by ADP. These results indicate that the Ca(2+)-ATPase described in this paper is the enzyme which drives active transport of Ca(2+) at the plasma membrane of higher plants.

Journal Article↗

PubMed1987

The Ca-Transport ATPase of Plant Plasma Membrane Catalyzes a nH/Ca Exchange.

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca(2+) upon addition of MgATP. MgATP-dependent Ca(2+) uptake co-migrates with the plasma membrane H(+)-ATPase on a sucrose gradient. Ca(2+) uptake is insensitive to oligomycin, inhibited by vanadate (IC(50) 40 micromolar) and erythrosin B (IC(50) 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca(2+) uptake is insensitive to protonophores. These results indicate that Ca(2+) transport in these microsomal vesicles is catalyzed by a Mg(2+)-dependent ATPase localized on the plasma membrane. Ca(2+) strongly reduces DeltapH generation by the plasma membrane H(+)-ATPase and increases MgATP-dependent membrane potential difference (Deltapsi) generation. These effects of Ca(2+) on DeltapH and Deltapsi generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca(2+) uptake into plasma membrane vesicles. The Ca(2+)-induced increase of Deltapsi is collapsed by permeant anions, which do not affect Ca(2+)-induced decrease of DeltapH generation by the plasma membrane H(+)-ATPase. The rate of decay of MgATP-dependent DeltapH, upon inhibition of the plasma membrane H(+)-ATPase, is accelerated by MgATP-dependent Ca(2+) uptake, indicating that the decrease of DeltapH generation induced by Ca(2+) reflects the efflux of H(+) coupled to Ca(2+) uptake into plasma membrane vesicles. It is therefore proposed that Ca(2+) transport at the plasma membrane is mediated by a Mg(2+)-dependent ATPase which catalyzes a nH(+)/Ca(2+) exchange.

Journal Article↗

PubMed1986

H-pumping driven by the plasma membrane ATPase in membrane vesicles from radish: stimulation by fusicoccin.

The 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 DeltapH probe acridine orange. Fusicoccin stimulated Mg:ATP-dependent H(+)-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 H(+)-pumping were completely inhibited by vanadate and almost unaffected by nitrate. Fusicoccin did not change membrane permeability to protons and fusicoccin-induced stimulation of Mg:ATP-dependent H(+)-pumping was not affected by changes in the buffer capacity of the incubation medium. Deacetylfusicoccin stimulated H(+)-pumping as much as fusicoccin, while the physiologically inactive derivative 8-oxo-9-epideacetylfusicoccin did not. Stimulation of H(+)-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(max) of the plasma membrane H(+)-ATPase rather than a decrease in its apparent K(m) for Mg:ATP.

Journal Article↗

PubMed1985

Fusicoccin stimulates the H+-ATPase of plasmalemma in isolated membrane vesicles from radish.

The effect of fusicoccin on the plasmalemma H+-ATPase has been investigated in a membrane fraction from 24 h old radish seedlings, in which Mg:ATP-dependent H+-transport is mediated only by the plasmalemma H+-ATPase. Fusicoccin stimulated the plasmalemma H+-ATPase - i.e. Mg:ATP-dependent intravesicular acidification, hyperpolaryzation of delta psi and ATPase activity -, when these activities were measured at the physiologically relevant pHs of 7.3 to 7.6. No effect of FC on the plasmalemma H+-ATPase was evident at pH 6.6.

Glycosides↗

PubMed1985

Electrogenic transport of protons driven by the plasma membrane ATPase in membrane vesicles from radish : biochemical characterization.

Mg:ATP-dependent H(+) pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Delta psi). DeltapH was measured as the decrease of absorbance of Acridine orange and Delta psi as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Delta pH and Delta psi generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Delta pH generation is not inhibited by NO(3) (-). These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H(+)-ATPases. Both intravesicular acidification and Delta psi generation are influenced by anions: Delta pH increases and Delta psi decreases following the sequence SO(4) (2-), Cl(-), Br(-), NO(3) (-). ATP-dependent H(+) pumping strictly requires Mg(2+). It is very specific for ATP (apparent K(m) 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Delta pH generation is inhibited by CuSO(4) and diethylstilbestrol as well as vanadate. Delta pH generation is specificially stimulated by K(+) (+ 80%) and to a lesser extent by Na(+) and choline (+28% and +14%, respectively). The characteristics of H(+) pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.

Journal Article↗

PubMed1981

Evidence for an electrogenic ATPase in microsomal vesicles from pea internodes.

The transmembrane electropotential of microsomal vesicles from pea internode segments, monitored by equilibrium distribution of the permeant anion SCN-, is strongly hyperpolarized when ATP is present in the incubation medium. The stimulation of SCN- uptake by ATP is rather specific with respect to the other nucleoside di- and triphosphates tested: ADP, GTP, CTP and UTP. ATP-stimulated SCN- uptake is strongly inhibited by ATPase inhibitors such as p-chloromercuribenzenesulphonate and N,N'-dicyclohexylcarbodiimide and by 2.5% toluene/ethanol (1 : 4, v/v), the latter being a treatment which makes the vesicles permeable. On the contrary, oligomycin is almost ineffective in influencing ATP-induced SCN- uptake. The proton conductor carbonyl cyanide p-trifluoromethoxyphenylhydrazone strongly inhibits ATP-stimulated SCN- uptake. The effect of ATP on SCN- uptake depends on the pH of the medium, the maximum being reached at about pH 7.0. These data support the view that microsomal fractions from pea internodes contain membrane vesicles endowed with a membrane-bound ATPase coupling ATP hydrolysis to electrogenic transport of ions, probably H+.

Adenosine Triphosphatases↗

PubMed1980

Relationship between ATP Level and Activity of Fusicoccin-stimulated H/K-Exchange System in Plant Tissues.

The treatment with fusicoccin causes a slight but significant decrease (about 15%) in the ATP level in pea-internode and maize-coleoptile segments. This decrease is detectable within 15 minutes and is accompanied by a parallel increase in O(2) uptake. Sodium azide inhibits O(2) uptake and completely blocks the stimulation of O(2) uptake by fusicoccin in both pea and coleoptile segments. Benzohydroxamic acid does not affect either basal or fusicoccin-induced O(2) uptake in maize-coleoptile sections. The drop of ATP level induced by various treatments (sodium arsenate, 2-deoxyglucose, limiting O(2), and anaerobiosis) is accompanied by a parallel inhibition of K(+) uptake in maize coleoptiles treated with or without fusicoccin. These results are consistent with the hypothesis that ATP is the energy source for the fusicoccin-activated H(+)/K(+)-exchange system.

Journal Article↗

PubMed1975