Effects of salt-adaptation and salt-stress on extracellular acidification and microsome phosphohydrolase activities in tomato cell suspensions

The effects of NaCl-adaptation and NaCl-stress on in vivo H⁺ extrusion and microsomal vanadate- and bafilomycin-sensitive ATPase and PPase activities were studied in tomato cell suspensions. Acidification of the external medium by 50 mM NaCl-adapted and non-adapted (control) tomato cells was similar. Extracellular acidification by both types of cells during the first hour of incubation with 2 μM fusicoccin (FC) in the presence of 100 mM NaCl was lightly increased while in the presence of 100 mM KCl it was increased by 3 (control)- and 6.5 (adapted)-fold. Extracellular alkalinization after 2 h of cell incubation in 100 mM NaCl indicated the possibility that a Na⁺/H⁺ exchange activity could be operating in both types of cells. Moreover, acidification induced by adding 100 mM NaCl + FC to non-adapted cells was relatively less affected by vanadate than that induced by 5 mM KCl + FC, which suggested that salt stress could induce some component other than H⁺ extrusion by H⁺-ATPase. In addition, no differences were observed in microsomal vanadate-sensitive ATPase activity among control, NaCl-adapted and NaCl-stressed cells, while K⁺-stimulated H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities were higher in microsomes from NaCl-adapted than in those from control cells. Likewise, the stimulation of in vivo H⁺ extrusion in NaCl adapted cells under NaCl or KCl stress in the presence of FC occurred with an inhibition of H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities and without changes in the vanadate-sensitive H⁺-ATPase activity. These results suggest that the stimulation of tonoplast proton pumps in NaCl-adapted cells, without changes in plasmalemma H⁺-ATPase, could serve to energize Na⁺ efflux across the plasmalemma and Na⁺ fluxes into vacuoles catalyzed by the Na⁺/H⁺ antiports. This revised version was published online in June 2006 with corrections to the Cover Date.     

Presence of Na+-Stimulated P-Type ATPase in the Membrane of a Facultatively Anaerobic Alkaliphile, Exiguobacterium aurantiacum

It was found that a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum, possesses a membrane-bound ATPase, which was activated specifically by Na⁺. The Na⁺-stimulated ATPase activity reached a maximum value at 200 mM NaCl. In the presence of 200 mM NaCl, the activity was drastically reduced by vanadate, a potent inhibitor of P-type ATPase, with a half-maximal inhibition at 1 μM. Incubation of the membranes with [γ-³²P]ATP followed by acidic lithium dodecyl sulfate–polyacrylamide gel electrophoresis demonstrated the existence of two phosphorylated intermediates with apparent molecular masses of 60 and 100 kDa. Only phosphorylation of the 100-kDa polypeptide was inhibited by vanadate. The membrane extract containing Na⁺-stimulated ATPase, when reconstituted into soybean phospholipid vesicles, exhibited ²²Na⁺ transport by the addition of ATP, which was inhibited by vanadate and gramicidin. It is likely that the Na⁺-stimulated ATPase belongs to P-type and is involved in Na⁺ transport. Received: 3 February 1999 / Accepted: 3 March 1999     

Relationship Between Effect of Ethanol on Proton Flux Across Plasma Membrane and Ethanol Tolerance, inPichia stipitis

Pichia stipitisefficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. InSaccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. InP. stipitisthe passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H⁺-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H⁺-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H⁺-extrusion. Comparison of the rates of H⁺-flux with the relatedin vitroH⁺-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) ofP. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H⁺-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grownP. stipitiscells.     

Effects of Intracellular Vanadate on Electrogenesis, Excitability and Cytoplasmic Streaming in Nitellopsis obtusa

Vanadate, which is known to inhibit the plasma membrane H^$-ATPaseof Neurospora, was applied intracellularly to internodal cellsof Nitellopsis by use of the intracellular perfusion technique.It inhibited electrogenesis and H^$-extrusion, evidence thatthe electrogenic pump of the Characeae plasmalemma is the H^$-extrudingone. The concentration of vanadate for the half-maximal inhibitionof the activity of the pump was 5 µM. The membrane potentialand H^$-extrusion occasionally recovered from vanadate inhibitionsfor reasons that are unknown. Membrane excitability, which isdependent on Mg?ATP, was not inhibited by vanadate, which suggeststhat the ATPase involved with membrane excitability differsfrom that of the H^$ pump. Cytoplasmic streaming took place evenwhen the cell was perfused with the medium containing 1 mM vanadate,which indicates that the vanadate-insensitive actomyosin systemis concerned with the motive force generation of the streaming. (Received August 27, 1981; Accepted April 13, 1982)     

Effect of phosphocreatine on H+ extrusion,pHi and dimorphism in Candida albicans

Candida albicans is an opportunistic pathogen. Its proliferation in human hosts is believed to be controlled by immunologic mechanisms. The plasma membrane of the fungus possesses an H(+)-ATPase (PM-ATPase) which actively extrudes protons to generate an electrochemical gradient which is used in co-transport of nutrients. This ATPase is associated with the growth, dimorphism and pathogenicity of the fungus. The physiological concentration of phosphocreatine (PCr) is 20-35 mM in skeletal muscles. H(+)-extrusion in Candida cells was strongly inhibited by PCr; 44% at 20 mM and 69% at 40 mM. H(+)-extrusion was stimulated 6.2-fold in the presence of 10 mM glucose. This glucose stimulated extrusion was inhibited significantly by PCr; 36% at 20 mM and 53% at 40 mM. The intracellular pH pattern of cells destined to differentiate was greatly altered in the presence of PCr. Evagination time for control cells was between 90-120 min. PCr, delayed dimorphism, reduced the population of cells differentiating to hyphae and also reduced the length of hyphae after each time interval. Only 60% differentiation was observed with 10 mM PCr and 40% for higher PCr concentration even after 210 min. Direct interaction of PM-ATPase and PCr has been demonstrated by difference spectrum measurement employing stopped flow spectrophotometer. It can be concluded that PCr may be playing a significant role in checking growth and pathogenesis of C. albicans.     

Effect of Mg2+, tritorn X-100 and temperature on basal and FC- stimulated plasma membrane ATPase activity

H⁺-pumping driven by the plasma membrane H⁺-ATPase in membrane vesicles from 24-hour-old radish seedlings is stimulated by pretreatment of the membranes with fusicoccin (FC) (Rasi-Caldogno et al., Plant Physiol., 82 (1986) 121).FC-pretreatment stimulates also the ATPase activity, but to a lesser extentthan H⁺-pumping. More than 80% of the ATPase activity is inhibited by 100 μM vanadate or by 3 mM Ca²⁺.Preincubation of diluted membranes in the presence of 5 mM MgSO₄ without ATP lowers both ATPase and H⁺-pumping activity by 20—30% without affecting FC-stimulated activities (i.e. the differences between FC-treated samples and the controls).After preincubation with MgSO₄, ATPase activity of membranes pretreatedwith or without FC is delivery affected by Triton X-100 and by temperature: Triton X-100 activates FC-stimulated ATPase more than that of the controls and an increase of temperature (between 13 and 33°C) enhances ATPase activity of the controls more than the FC-stimulated one.These results have been interpreted as suggesting that, while H⁺-pumping in this membrane fraction is driven only by the plasma membrane H⁺-ATPase, ATP-hydrolysis is catalyzed by two different enzymes (or forms of the same enzxxyme) diversely sensitive to FC, Triton X-100 and temperature and possibly diversely involved in H⁺-pumping.     

Vanadate inhibition of stomatal opening in epidermal peels of Commelina communis

An H⁺ ATPase at the plasma-membrane of guard cells is thought to establish an electrochemical gradient that drives K⁺ and Cl^– uptake, resulting in osmotic swelling of the guard cells and stomatal opening. There are, however, conflicting results regarding the effectiveness of the plasma-membrane H⁺-ATPase inhibitor, vanadate, in inhibiting both H⁺ extrusion from guard cells and stomatal opening. We found that 1 mM vanadate inhibited light-stimulated stomatal opening in epidermal peels of Commelina communis L. only at KCl concentrations lower than 50 mM. When impermeant n-methylglucamine and HCl (pH 7.2) were substituted for KCl, vanadate inhibition was still not observed at total salt concentrations50 mM. In contrast, in the absence of Cl^–, when V₂O₅ was used to buffer KOH, vanadate inhibition of stomatal opening occurred at K⁺ concentrations as high as 70 mM. Partial vanadate inhibition was observed in the presence of the impermeant anion, iminodiacetic acid (100 mM KHN(CH₂CO₂H)₂). These results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect. In support of this hypothesis, an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially prevented vanadate inhibition of stomatal opening. Other anion-uptake inhibitors (1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid, 200 M Zn²⁺) were not effective. Decreased vanadate inhibition at high Cl^–/vanadate ratios may result from competition between vanadate and Cl^– for uptake. Unlike metabolic inhibitors, vanadate did not affect the extent of stomatal closure stimulated by darkness, further indicating that the observed action of vanadate represents a specific inhibition of the guard-cell H⁺ ATPase.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - FC fusicoccin - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid We thank Drs. R.T. Leonard (University of California, Riverside, USA) and K.A, Rubinson (Yellow Springs, Oh., USA) for helpful comments on the research, Janet Sherwood (Harvard University) for excellent plant care, and Angela Ciamarra, Anne Gershenson, Gustavo Lara (Harvard University) and Orit Tal (Hebrew University) for valuable technical assistance. This research was supported by a grant from the National Science Foundation (DCB-8904041) to S.M.A.     

ATP-induced sucrose efflux from red-beet tonoplast vesicles

 Sucrose efflux from the vacuole of mobilizing red-beet (Beta vulgaris L.) hypocotyl cells was investigated using purified tonoplast vesicles. Tonoplast vesicle purity was assured by the immunoreactivity to antibodies raised against the vacuolar ATPase and by the strong inhibition exhibited by the H⁺-ATPase to bafilomycin-A and NO₃ ⁻. Inhibition of the H⁺-ATPase by vanadate and azide was negligible. Sucrose was loaded into tonoplast vesicles by using the pH-jump method of energization. Addition of ATP to sucrose-loaded vesicles in the presence of bafilomycin-A resulted in efflux of a significant amount of sucrose. During ATP-induced sucrose efflux, bafilomycin-insensitive ATPase activity increased significantly with no increase in H⁺-translocating activity. The additional bafilomycin-A insensitive ATPase activity observed in sucrose-loaded vesicles was completely inhibited by vanadate as was the efflux of sucrose. Similar to vanadate, thapsigargin was also inhibitory to sucrose efflux and to the bafilomycin-A insensitive ATPase activity. The data indicate that vacuolar sucrose can be actively mobilized by a specific ATP-dependent efflux mechanism. Received: 12 October 1999 / Accepted: 18 November 1999     

H-pumping driven by the vanadate-sensitive ATPase in membrane vesicles from corn roots

The initial rate of quenching of quinacrine fluorescence was used to monitor Mg:ATP-dependent H⁺-pumping in membrane vesicles from corn (Zea mays L. cv WF9 × MO17) roots and obtain a preparation in which vanadate-sensitive H⁺-pumping could be observed. Separation of membranes on a linear sucrose density gradient resulted in two distinct peaks of H⁺-pumping activity: a major one, at density 1.11 grams per cubic centimeter, was sensitive to NO₃⁻ and resistant to vanadate, while a minor one, at density 1.17 grams per cubic centimeter, was substantially resistant to NO₃⁻ and sensitive to vanadate. A membrane fraction enriched in the vanadate-sensitive H⁺-pump could be obtained by washing microsomes prepared in the presence of 10% glycerol with 0.25 molar KI. The kinetics of inhibition of H⁺-pumping by vanadate in this membrane preparation indicated that most of the H⁺-pumping activity in this fraction is sensitive to inhibition by vanadate, 50% inhibition being reached at about 60 micromolar vanadate. This value is fairly close to that observed for inhibition by vanadate of the ATPase activity in similar experimental conditions (40 micromolar). The inhibitor sensitivity, divalent cation dependence, pH optimum (6.5), and K_m for ATP (0.7 millimolar) of the H⁺-pumping activity match quite closely those reported for the plasma membrane ATPase of corn roots and other plant materials.     

HELIOTROPIC LEAF MOVEMENT RESPONSE TO H+/ATPase activation,H+/ATPase inhibition,AND K+ CHANNEL INHIBITION IN VIVO

The pulvinus, located at the base of soybean leaflets, is both the light perception and motor organ for heliotropic leaf movements. Our objective was to investigate the role of plasma membrane H⁺/ATPase and TEA-sensitive K⁺ channels in mediating pulvinar response to light. The plasma membrane H+/ATPase activator, fusicoccin, plasma membrane H⁺/ATPase inhibitors, vanadate and erythrosin-B, and the K⁺ channel blocker TEA were introduced to the intact pulvinus through the transpiration stream. The pulvinus was illuminated by a vertical light beam of 1,400 μmol m^-2 s^-1 to stimulate leaf movement. Leaf orientation was measured every 5 min for 60 min of illumination. All compounds tested inhibited pulvinar bending, but concentration and uptake time required for inhibition varied: 12.5 μM fusicoccin reduced leaf movement after 3 hr uptake, 2 mM vanadate reduced leaf movement after 6 hr uptake, 100 μM erythrosin-B reduced leaf movement after 3 hr uptake, and 15 mM TEA reduced leaf movement after 6 hr uptake. In all cases final leaf angle was reduced by higher concentrations and/or increased time for uptake of the chemical into the pulvinus. Results support the hypothesis that the proximal mechanism of heliotropic movement is similar to that of nyctinastic movements.     

Proton-pumping adenosine triphosphatase in membrane vesicles of tobacco callus: Sensitivity to vanadate and K+

H⁺-pumping adenosinetriphosphatases (ATPases, EC 3.6.1.3) were demonstrated in sealed microsomal vesicles of tobacco callus. Quinacrine fluorescence quenching was induced specifically by MgATP and stimulated by EGTA and Cl^?. Fluorescence quenching reflected a relative measure of pH gradient formation (inside acid), as it could be reversed by gramicidin (an H⁺/cation conductor) or 10 mM NH₄Cl (an uncoupler). H⁺ pumping was inhibited by tributyltin (an ATPase inhibitor) and sodium vanadate, but it was insensitive to oligomycin or fusicoccin. The vanadate concentration required to inhibit pH gradient formation was similar to that needed to inhibit KCl-stimulated Mg²⁺-ATPase activity and generation of a membrane potential (measured by ATP-dependent ³⁵SCN^? uptake). About 45% of all three activities (ATPase, pH gradient, membrane potential generation) were vanadate-insensitive, supporting the idea that non-mitochondrial membranes of plants have at least two types of electrogenic H⁺ pump.A vanadate-insensitive, H⁺-pumping ATPase previously shown by methylamine accumulation was characterized to be anion-sensitive and possibly enriched in vacuolar membranes (Churchill, K.A. and Sze, H. (1983) Plant Physiol. 71, 610–617). Yet, pH gradient formation determined by quinacrine fluorescence quenching was decreased by monovalent cations with a sequence K⁺, Rb⁺, Na⁺ > Cs⁺,Li⁺> choline, bisTris-propane. Since K⁺ stimulated ATPase activity more than Bistris-propane, K⁺ appeared to collapse formation of the pH gradient by an H⁺/K⁺ countertransport. The sensitivity to vanadate and K⁺ provides evidence that the plasma-membrane ATPase is an electrogenic H⁺ pump.     

Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris

Summary A vanadate-sensitive H⁺-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H⁺-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H⁺ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H⁺ transport activity was inhibited half-maximally between 1 and 5 M vanadate (Na₃VO₄) and nearly abolished by 100 M vanadate. ATPase activity of native plasma membrane showed aK _i for vanadate inhibition of 9.5 M, and was inhibited up to 80% by 15 to 20 M vanadate (Na₃VO₄). ATPase activity of the reconstituted vesicles showed aK _i of 2.6 M for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme.     

Effects of Two Plasmalemma ATPase Inhibitors on H+ Extrusion and Intracellular pH in Elodea densa Leaves

Beffagna, N. and Romani, G. 1988. Effects of two plasmalemmaATPase inhibitors on H⁺ extrusion and intracellular pH in Elodeadensa leaves.—J. exp. Bot. 39: 1033–1043. Elodea leaves in the dark show very little exchange of H⁺ withthe medium in the external pH range between 5.0 and 6.0. Thepresence of fusicoccin and potassium in the medium markedlystimulates H⁺ extrusion. Fusicoccin- and K⁺ -induced H⁺ extrusionis inhibited by either erythrosin B (EB) or Na-orthovanadate,two inhibitors of H⁺ transporting plasma membrane ATPase. EBcompletely inhibits it from the first 30 min of treatment, whensupplied at pH 5.5 at a concentration of 30 mmol m^–3.Vanadate also inhibits H⁺ extrusion, this effect becoming evidentonly after 45 min of treatment. After this time inhibition iscomplete with 250 mmol m^–3 vanadate but only partial forlower concentrations. In the presence of either inhibitor the intracellular pH, measuredas cell sap pH, is significantly lowered. When the intracellularpH changes are determined on vacuole and, separately, on cytoplasmby the weak acid and base distribution method, acidificationof both compartments is found to accompany the blocking of H⁺extrusion by either of the inhibitors. Key words: Intracellular pH, vanadate, erythrosin B, H⁺pumping     

Effects of vanadate on the plasma membrane ATPase of red beet and corn

The effect of vanadate on the plant plasma membrane ATPase were investigated in plasma membrane fractions derived from corn roots (Zea mays L.) and red beets (Beta vulgaris L.). The K_i for vanadate inhibition of the plasma membrane ATPase from corn roots and red beets was between 6 and 15 micromolar vanadate. In both membrane fractions, 80% to 90% of the total ATPase was inhibited at vanadate concentrations below 100 micromolar. Vanadate inhibition was optimal at pH 6.5, enhanced by the presence of K⁺, and was partially reversed by 1 millimolar EDTA. The Mg:ATP kinetics for the plasma membrane ATPase were hyperbolic in both the absence and presence of vanadate. Vanadate decreased both the K_m and V_max of the red beet plasma membrane ATPase, indicating that vanadate inhibits the ATPase uncompetitively. These results indicate many similarities with respect to vanadate inhibition between the plant plasma membrane ATPase and other major iontranslocating ATPases from fungal and animal cells. The high sensitivity to vanadate reported here, however, differs from other reports of vanadate inhibition of the plant plasma membrane ATPase from corn, beets, and in some instances oats.     

Erythrosin B as an effective inhibitor of electrogenic H+ extrusion

Abstract In 24-h-genninaled radish seedlings erythrosin B (EB), an effective inhibitor of microsomal as well as of partially purified vanadate-sensitive ATPase markedly inhibited the basal and the FC-stimulated proton extrusion, and induced a rapid depolarization of FC-hyperpolarized trans-membrane electric potential (PD) without causing any significant change of ATP level. The effects of EB on H⁺ extrusion were partially additive with those of vanadatc, another inhibitor of plasma membrane H⁺-ATPase. These effects are interpreted as due to a direct inhibition by EB on plasma membrane H⁺-ATPase involved in H⁺ electrogenic transport in the higher plants.     

Plasmalemma redox activity and h extrusion: I. Activation of the h-pump by ferricyanide-induced potential depolarization and cytoplasm acidification

Ferricyanide reduction by Elodea densa leaves, in the dark, is associated with: (a) acidification of the medium; (b) decrease (about 0.2-0.3 units) of intracellular pH (measured in cell sap, cytoplasm, and vacuole); (c) depolarization of the transmembrane potential; (d) net efflux of K⁺ to the medium. Ferricyanide-induced acid secretion is markedly increased by the presence of fusicoccin (FC), and this effect is severely inhibited by the proton pump inhibitors erythrosine B and vanadate. In the presence of ferricyanide FC-induced H⁺ extrusion no longer requires the presence of K⁺ in the medium. The (ferricyanide reduced)/(H⁺ extruded) ratio varies from about 2, in the absence of FC, to about 1 when the toxin is present, and to more than 4, when ATP-driven H⁺ extrusion is inhibited by erythrosine B or by vanadate. Fusicoccin markedly reduces K⁺ release to the medium. The ratio (ferricyanide reduced)/(H⁺ extruded + K⁺ released) approaches unity under all of the three conditions considered. These results indicate that ferricyanide reduction depends on a plasmalemma system transporting only electrons to the extracellular acceptor, with consequent potential depolarization and cytoplasm acidification. Most of the protons released in the cytoplasm would be secondarily extruded by the ATP-driven pump, stimulated by both intracellular acidification and depolarization. K⁺ efflux would depend on potential depolarization.     

Evidence that the partial resistance of the Arabidopsis 5-2 mutant to fusicoccin depends on a decreased capacity of the H+ pump to respond to activating factors

Measurements of H⁺ extrusion activity K⁺ influx, and Es bm in 3-d-old seedlings of the 5-2 mutant of Arabidopsis thaliana (which is partially insensitive to fusicoccin) showed the following, (i) The reduced response of 5-2 to fusicoccin (FC) does not depend on the penetration of FC to its site of action, or on decreased affinity of the FC receptor, (ii) The reduced response of H⁺ and K⁺ transport to FC does not depend on an impairment of the K⁺ absorption system, (iii) The mutation can influence the H⁺ extrusion system independently of the presence of FC. In the presence of factors other than FC known to activate the plasma membrane H⁺-ATPase (e.g. a cytosol-acidifying treatment), the response in 5-2 is about 50% lower than in wt. (iv) When both genotypes grow in optimal conditions, the rate of fresh weight increase and stem elongation is higher in wt than 5-2. These data indicate that the 5-2 mutation affects some intrinsic component of the H⁺-extrusion machinery, the limiting effect of which becomes considerable when either the physiological or the experimental conditions induce a high level of proton pump activity. An alteration either of the ATPase itself or of a factor controlling its activity is compatible with our observations.     

Separation of two types of electrogenic h-pumping ATPases from oat roots

Microsomal vesicles of oat roots (Avena sativa var Lang) were separated with a linear dextran (0.5-10%, w/w) or sucrose (25-45%, w/w) gradient to determine the types and membrane identity of proton-pumping ATPases associated with plant membranes. ATPase activity stimulated by the H⁺/K⁺ exchange ionophore nigericin exhibited two peaks of activity on a linear dextran gradient. ATPase activities or ATP-generated membrane potential (inside positive), monitored by SCN⁻ distribution, included a vanadate-insensitive and a vanadate-sensitive component. In a previous communication, we reported that ATP-dependent pH gradient formation (acid inside), monitored by quinacrine fluorescence quenching, was also partially inhibited by vanadate (Churchill and Sze 1983 Plant Physiol 71: 610-617). Here we show that the vanadate-insensitive, electrogenic ATPase activity was enriched in the low density vesicles (1-4% dextran or 25-32% sucrose) while the vanadate-sensitive activity was enriched at 4% to 7% dextran or 32% to 37% sucrose. The low-density ATPase was stimulated by Cl⁻ and inhibited by NO⁻₃ or 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS). The distribution of Cl⁻-stimulated ATPase activity in a linear dextran gradient correlated with the distribution of H⁺ pumping into vesicles as monitored by [¹⁴C]methylamine accumulation. The vanadate-inhibited ATPase was mostly insensitive to anions or DIDS and stimulated by K⁺. These results show that microsomal vesicles of plant tissues have at least two types of electrogenic, proton-pumping ATPases. The vanadate-insensitive and Cl⁻-stimulated, H⁺-pumping ATPase may be enriched in vacuolar-type membranes; the H⁺-pumping ATPase that is stimulated by K⁺ and inhibited by vanadate is most likely associated with plasma membrane-type vesicles.     

Role of the Plasmalemma H-ATPase in Pseudomonas syringae-Induced K/H Exchange in Suspension-Cultured Tobacco Cells

Activation of a host plasma membrane K⁺ efflux/net H⁺ uptake exchange by pathogenic pseudomonads plays an important role in the development of hypersensitivity in tobacco (Nicotiana tabacum). Involvement of the plasmalemma H⁺-pumping ATPase in this response was investigated. The exchange response of suspension-cultured tobacco cells to Pseudomonas syringae pv syringae was reduced 90% or more by ATPase inhibitors including vanadate, N-ethylmaleimide, and N,N′-dicyclohexylcarbodiimide. The exchange was also strongly inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone and by slightly alkaline external pH. Respiratory inhibitors such as oligomycin and sodium azide reduced the exchange by 50% to 75%, while glycolysis inhibitors such as sodium arsenite and sodium iodoacetate decreased exchange by approximately 90%. These results suggest that plasmalemma H⁺-ATPase activity is required for the exchange response and that this may reflect a requirement for a plasmalemma pH and/or electrical potential gradient.     

Proton Transport Activity of the Purified Vacuolar H-ATPase from Oats : Direct Stimulation by Cl

To determine whether the detergent-solubilized and purified vacuolar H⁺-ATPase from plants was active in H⁺ transport, we reconstituted the purified vacuolar ATPase from oat roots (Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP hydrolysis activity of the reconstituted ATPase was stimulated twofold by gramicidin, suggesting that the enzyme was incorporated into sealed proteoliposomes. Acidification of K⁺-loaded proteoliposomes, monitored by the quenching of acridine orange fluorescence, was stimulated by valinomycin. Because the presence of K⁺ and valinomycin dissipates a transmembrane electrical potential, the results indicate that ATP-dependent H⁺ pumping was electrogenic. Both H⁺ pumping and ATP hydrolysis activity of reconstituted preparations were completely inhibited by <50 nanomolar bafilomycin A₁, a specific vacuolar type ATPase inhibitor. The reconstituted H⁺ pump was also inhibited by N,N′-dicyclohexylcarbodiimide or NO₃⁻ but not by azide or vanadate. Chloride stimulated both ATP hydrolysis by the purified ATPase and H⁺ pumping by the reconstituted ATPase in the presence of K⁺ and valinomycin. Hence, our results support the idea that the vacuolar H⁺-pumping ATPase from oat, unlike some animal vacuolar ATPases, could be regulated directly by cytoplasmic Cl⁻ concentration. The purified and reconstituted H⁺-ATPase was composed of 10 polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H⁺ pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H⁺ translocation.