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 (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO₃⁻. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H⁺-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO₄²⁻, Cl⁻, Br⁻, NO₃⁻. ATP-dependent H⁺ pumping strictly requires Mg²⁺. It is very specific for ATP (apparent K_m 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO₄ and diethylstilbestrol as well as vanadate. Δ 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.     

Collapse of ATP-Induced pH Gradient by Sodium Ions in Microsomal Membrane Vesicles Prepared from Atriplex gmelini Leaves: Possibility of Na/H Antiport

Sealed microsomal membrane vesicles were prepared from leaves of a 250 millimolar NaCl-grown halophyte (Atriplex gmelini C. A. Mey). The vesicles exhibited ATP-dependent proton-transporting activity which was inhibited 60% by NO₃⁻ (50 millimolar) but not by vanadate (100 micromolar) and 23% by oligomycin (10 micrograms per milliliter), suggesting that tonoplast-derived vesicles were the major constituents of the preparation. The pH gradient established by the vesicles by ATP in the presence of oligomycin collapsed upon the addition of Na⁺ salts. The vesicles took up Na⁺ ions in the presence of ATP and this activity was canceled by gramicidin. These results suggest that Na⁺ ions were taken up by the vesicles via a Na⁺-specific uptake system, possibly a Na⁺/H⁺ antiport.     

Studies on H-Translocating ATPases in Plants of Varying Resistance to Salinity : I. Salinity during Growth Modulates the Proton Pump in the Halophyte Atriplex nummularia

Membrane vesicles were isolated from the roots of the halophyte Atriplex nummularia Lindl. H⁺-translocating Mg²⁺-ATPase activity was manifested by the establishment of a positive membrane potential (measured as SCN⁻ accumulation); and also by the establishment of a transmembrane pH gradient (measured by quinacrine fluorescence quenching). H⁺-translocation was highly specific to ATP and was stable to oligomycin. Growing the plants in the presence of 400 millimolar NaCl doubled the proton-translocating activity per milligram of membrane protein and otherwise modulated it in the following ways. First, the flat pH profile observed in non-salt-grown plants was transformed to one showing a peak at about pH 6.2. Second, the lag effect observed at low ATP concentration in curves relating SCN⁻ accumulation to ATP concentration was abolished; the concave curvature shown in the double reciprocal plot was diminished. Third, sensitivity to K-2 (N-morpholino)ethanesulfonic acid stimulation was shown in salt-grown plants (about 40% stimulation) but was absent in non-salt-grown plants. Fourth, the KCl concentration bringing about 50% dissipation of ATP-dependent SCN⁻ accumulation was 20 millimolar for salt-grown plants and 50 millimolar for non-salt-grown plants. Vanadate sensitivity was shown in both cases. No clear NO₃⁻ inhibition was observed.     

Studies on H-Translocating ATPases in Plants of Varying Resistance to Salinity : II. K Strongly Promotes Development of Membrane Potential in Vesicles from Cotton Roots

Mg²⁺-ATP-dependent H⁺-translocation has been studied in membrane vesicles derived from the roots of Gossypium hirsutum L. var. Acala San Jose 2. Establishment of a positive membrane potential was followed by measuring SCN⁻ accumulation; establishment of ΔpH across the vesicle membranes by measuring quinacrine fluorescence quenching. High specificity for ATP was shown, and H⁺-translocation was oligomycin stable. The pH profile for H⁺-translocation showed an optimum at 5.5. The relationship between SCN⁻ accumulation and ATP concentration was approximately Michaelian; the apparent K_m was 0.7 millimolar. K-2-(N-morpholino)ethanesulfonic acid strongly promoted ATP-dependent SCN⁻ uptake (up to 180% stimulation). The effect was not given by Na-Mes. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone totally inhibited SCN⁻ accumulation, both in the presence and absence of K-2(N-morpholino)ethanesulfonic acid. Vanadate at 200 micromolar inhibited SCN⁻ uptake by about 10 to 40% in the absence of K⁺, but more strongly in its presence (about 60%). NO₃⁻ at 100 millimolar inhibited initial rate of quinacrine quenching by about 25%. The NO₃⁻ insensitive fraction was activated by K⁺; and inhibited by 200 micromolar vanadate to about 40%, provided K⁺ was present. Saline conditions during the growth of the plants had no appreciable effect on the observed characteristics of H⁺-translocation.     

Potential-dependent anion transport in tonoplast vesicles from oat roots

Potential-dependent anion movement into tonoplast vesicles from oat roots (Avena sativa L. var Lang) was monitored as dissipation of membrane potentials (Δψ) using the fluorescence probe Oxonol V. The potentials (positive inside) were generated with the H⁺-pumping pyrophosphatase, which is K⁺ stimulated and anion insensitive. The relative rate of ΔΨ dissipation by anions was used to estimate the relative permeabilities of the anions. In decreasing order they were: SCN⁻ (100) > NO₃⁻ (72) = Cl⁻ (70) > Br⁻ (62) > SO₄²⁻ (5) = H₂PO₄⁻ (5) > malate (3) = acetate (3) > iminodiacetate (2). Kinetic studies showed that the rate of Δψ dissipation by Cl⁻ and NO₃⁻, but not by SCN⁻, was saturable. The K_m values for Cl⁻ and NO₃⁻ uptake were about 2.3 and 5 millimolar, respectively, suggesting these anions move into the vacuole through proteinaceous porters. In contrast to a H⁺-coupled Cl⁻ transporter on the same vesicles, the potential-dependent Cl⁻ transport was insensitive to 4,4′-diisothiocyano-2,2′-stilbene disulfonate. These results suggest the existence of at least two different mechanisms for Cl⁻ transport in these vesicles. The potentials generated by the H⁺-translocating ATPase and H⁺-pyrophosphatase were nonadditive, giving support to the model that both pumps are on tonoplast vesicles. No evidence for a putative Cl⁻ conductance on the anion-sensitive H⁺-ATPase was found.     

Evidence for a highly specific k/h antiporter in membrane vesicles from oil-seed rape hypocotyls

We present evidence strongly suggesting that a proton gradient (acid inside) is used to drive an electroneutral, substrate-specific, K⁺/H⁺ antiport in both tonoplast and plasma membrane-enriched vesicles obtained from oilseed rape (Brassica napus) hypocotyls. Proton fluxes into and out of the vesicles were monitored both by following the quenching and restoration of quinacrine fluorescence (indicating a transmembrane pH gradient) and of oxonol V fluorescence (indicating membrane potential.) Supply of K⁺ (with Cl⁻ or SCN⁻) after a pH gradient had been established across the vesicle membrane by provision of ATP to the H⁺-ATPase dissipated the transmembrane pH gradient but did not depolarize the positive membrane potential. Evidence that the K⁺/H⁺ exchange thus indicated could not be accounted for by mere electric coupling included the findings that, first, no positive potential was generated when KSCN or KCl was supplied, even in the absence of 100 millimolar Cl⁻ and, second, efflux of K⁺ from K⁺-loaded vesicles drives intravesicular accumulation of H⁺ against the electrochemical potential gradient. Neither was the exchange due to competition between K⁺ and quinacrine for membrane sites, nor to inhibition of the H⁺-ATPase. Thus, it is likely that it was effected by a membrane component. The exchanger utilized primarily K⁺ (at micromolar concentrations); Na⁺/H⁺ antiport was detected only at concentrations two orders of magnitude higher. Rb⁺, Li⁺, or Cs⁺ were ineffective. Dependence of tonoplast K⁺/H⁺ antiport on K⁺ concentration was complex, showing saturation at 10 millimolar K⁺ and inhibition by concentrations higher than 25 millimolar. Antiport activity was associated both with tonoplast-enriched membrane vesicles (where the proton pump was inhibited by more than 80% by 50 millimolar NO₃⁻ and showed no sensitivity to vanadate or oligomycin) and with plasma membrane-enriched fractions prepared by phase separation followed by separation on a sucrose gradient (where the proton pump was vanadate and diethylstilbestrol-sensitive but showed no sensitivity to NO₃⁻ or oligomycin). The possible physiological role of such a K⁺/H⁺ exchange mechanism is discussed.     

Anion-Sensitive, H-Pumping ATPase of Oat Roots : Direct Effects of Cl, NO(3), and a Disulfonic Stilbene

To understand the mechanism and molecular properties of the tonoplast-type H⁺-translocating ATPase, we have studied the effect of Cl⁻, NO₃⁻, and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H⁺-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H⁺-pumping ATPase generates a membrane potential (Δψ) and a pH gradient (ΔpH) that make up two interconvertible components of the proton electrochemical gradient (μh⁺). A permeant anion (e.g. Cl⁻), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([¹⁴C]thiocyanate distribution) and stimulated formation of a pH gradient ([¹⁴C]methylamine distribution). However, Cl⁻-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl⁻ independent of the proton electrochemical gradient. Unlike the plasma membrane H⁺-ATPase, the Cl⁻-stimulated ATPase was inhibited by NO₃⁻ (a permeant anion) and by DIDS. In the absence of Cl⁻, NO₃⁻ decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO₃⁻ of Cl⁻-stimulated pH gradient formation and Cl⁻-stimulated ATPase activity was noncompetitive. In the absence of Cl⁻, DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl⁻-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H⁺-ATPase by NO₃⁻ or DIDS suggest that the vanadate-insensitive H⁺-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion-sensitive H⁺-ATPase has channels that conduct anions is yet to be established.     

Decrease of pH Gradients in Tonoplast Vesicles by NO(3) and Cl: Evidence for H-Coupled Anion Transport

Chloride or nitrate decreased a pH gradient (measured as [¹⁴C]methylamine accumulation) in tonoplast-enriched vesicles. The ΔpH decrease was dependent on the anion concentration. These effects are independent of the anion-sensitive H⁺-ATPase of the tonoplast, since the pH gradient (acid inside) was imposed artificially using a pH jump or a K⁺ gradient and nigericin. 4,4′-Diisothiocyano-2,2′-stilbene disulfonic acid partially prevented the decrease in pH gradient induced by Cl⁻. Two possible models to account for this anion-dependent decrease of ΔpH are: (a) H⁺ loss is accompanied by Cl⁻ or NO₃⁻ efflux from the vesicles via H⁺/anion symport systems on the tonoplast and (b) H⁺ loss is accompanied by Cl⁻ or NO₃⁻ uptake into the vesicles via H⁺/anion antiport systems. Depending on the requirements and conditions of the cell, these two systems would serve to either mobilize Cl⁻ and NO₃⁻ stored in the vacuole for use in the cytoplasm or to drive anions into the vacuole. Chloride or nitrate also decreased a pH gradient in fractions containing plasma membrane and Golgi, implying that these membranes may have similar H⁺-coupled anion transport systems.     

Effect of Ca and Calmodulin on DeltapH Formation in Tonoplast Vesicles from Corn Roots

The effects of calmodulin (CaM) on ATPase activity and ATP-dependent formation of a proton gradient (ΔpH) were studied in tonoplast membrane vesicles from corn (Zea mays L.) roots. At 0.6 micromolar, CaM stimulated ATPase activity by about 20% in the absence of an uncoupler, but by only 4% in its presence. Thus, the uncoupler-dependent increment of activity was decreased 30 to 45% by CaM. The formation of a proton gradient across the membrane vesicle, measured by quinacrine fluorescence quench, was inhibited about 20% by CaM. Its effect was additive to the effect of Ca²⁺ and was completely abolished by EGTA. These effects of CaM could be due to stimulation of H⁺ efflux or due to inhibition of the H⁺-ATPase. To distinguish between these possibilities, we examined the effect of CaM on dissipation of preformed ΔpH after the ATPase was inhibited. CaM stimulated the dissipation of a preformed ΔpH by 40% after the H⁺-ATPase was inhibited with NO₃⁻. This indicates that CaM facilitates the recycling of protons across the tonoplast membranes and does not regulate the H⁺-ATPase by direct inhibition.     

Anion-sensitive, h-pumping ATPase in membrane vesicles from oat roots

H⁺-pumping ATPases were detected in microsomal vesicles of oat (Avena sativa L. var Lang) roots using [¹⁴C]methylamine distribution or quinacrine fluorescent quenching. Methylamine (MeA) accumulation into vesicles and quinacrine quench were specifically dependent on Mg,ATP. Both activities reflected formation of a proton gradient (ΔpH) (acid inside) as carbonyl cyanide m-chlorophenylhydrazone, nigericin (in the presence of K⁺), or gramicidin decreased MeA uptake or increased quinacrine fluorescence. The properties of H⁺ pumping as measured by MeA uptake were characterized. The K_mapp for ATP was about 0.1 millimolar. Mg,GTP and Mg, pyrophosphate were 19% and 30% as effective as Mg,ATP. MeA uptake was inhibited by N,N′-dicyclohexylcarbodiimide and was mostly insensitive to oligomycin, vanadate, or copper. ATP-dependent MeA was stimulated by anions with decreasing order of potency of Cl⁻ > Br⁻ > NO₃⁻ > SO₄²⁻, iminodiacetate, benzene sulfonate. Anion stimulation of H⁺ pumping was caused in part by the ability of permeant anions to dissipate the electrical potential and in part by a specific requirement of Cl⁻ by a H⁺ -pumping ATPase. A pH gradient, probably caused by a Donnan potential, could be dissipated by K⁺ in the presence or absence of ATP. MeA uptake was enriched in vesicles of relatively low density and showed a parallel distribution with vanadate-insensitive ATPase activity on a continuous dextran gradient. ΔpH as measured by quinacrine quench was partially vanadate-sensitive. These results show that plant membranes have at least two types of H⁺ -pumping ATPases. One is vanadate-sensitive and probably enriched in the plasma membrane. One is vanadate-resistant, anion-sensitive and has many properties characteristic of a vacuolar ATPase. These results are consistent with the presence of electrogenic H⁺ pumps at the plasma membrane and tonoplast of higher plant cells.     

Electrostatic Changes in Lycopersicon esculentum Root Plasma Membrane Resulting from Salt Stress

Salinity-induced alterations in tomato (Lypersicon esculentum Mill. cv Heinz 1350) root plasma membrane properties were studied and characterized using a membrane vesicle system. Equivalent rates of MgATP-dependent H⁺-transport activity were measured by quinacrine fluorescence (ΔpH) in plasma membrane vesicles isolated from control or salt-stressed (75 millimolar salt) tomato roots. However, when bis-[3-phenyl-5-oxoisoxazol-4-yl] pentamethine was used to measure MgATP-dependent membrane potential (ΔΨ) formation, salt-stressed vesicles displayed a 50% greater initial quench rate and a 30% greater steady state quench than control vesicles. This differential probe response suggested a difference in surface properties between control and salt-stressed membranes. Fluorescence titration of vesicles with the surface potential probe, 8-anilino-1-napthalenesulphonic acid (ANS) provided dissociation constants (K_d) of 120 and 76 micromolar for dye binding to control and salt-stressed vesicles, respectively. Membrane surface potentials (Ψ_o) of−26.0 and −13.7 millivolts were calculated for control and salt-stressed membrane vesicles from the measured K_d values and the calculated intrinsic affinity constant, K_i. The concentration of cations and anions at the surface of control and salt-stressed membranes was estimated using Ψ_o values and the Boltzmann equation. The observed difference in membrane surface electrostatic properties was consistent with the measured differences in K⁺-stimulated kinetics of ATPase activity between control and salt-stressed vesicles and by the differential ability of Cl⁻ ions to stimulate H⁺-transport activity. Salinity-induced changes in plasma membrane electrostatic properties may influence ion transport across the plasma membrane.     

Na/H and k/h antiport in root membrane vesicles isolated from the halophyte atriplex and the glycophyte cotton

Proton fluxes have been followed into and out of membrane vesicles isolated from the roots of the halophyte Atriplex nummularia and the glycophyte Gossypium hirsutum, with the aid of the ΔpH probe [¹⁴C]methylamine. Evidence is presented for the operation of Na⁺/H⁺ and K⁺/H⁺ antiporters in the membranes of both plants. Cation supply after a pH gradient has been set up across the vesicle membrane (either as a result of providing ATP to the H⁺-ATPase or by imposing an artificial pH gradient) brings about dissipation of the ΔpH, but does not depolarize the membrane potential as observed in similar experiments, but in the absence of Cl⁻, using the ΔΨ probe SCN⁻. Cation/H⁺ exchange is thus indicated. This exchange is not due to nonspecific electric coupling, nor to competition for anionic adsorption sites on the membrane, nor to inhibition of the H⁺-ATPase; coupling of the opposed cation and H⁺ fluxes by a membrane component is the most likely explanation. Saturation kinetics have been observed for both Na⁺/H⁺ and K⁺/H⁺ antiport in Atriplex. Moreover, additive effects are obtained when Na⁺ is supplied together with saturating concentrations of K⁺, and vice versa, suggesting that separate antiporters for Na⁺ and for K⁺ may be operating. In the case of both Atriplex and Gossypium evidence was obtained suggesting the presence of antiporters in both plasmalemma and tonoplast.     

Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots

A H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H⁺-PPase catalyzed the electrogenic transport of H⁺ into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb⁺ = K⁺ > Cs⁺; but it was inhibited by Na⁺ or Li⁺. Maximum rates of transport required at least 20 millimolar K⁺ and the K_m for this ion was 4 millimolar. Fluoride inhibited both ΔpH formation and K⁺-dependent PPase activity with an I₅₀ of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H⁺-ATPase (e.g. a disulfonic stilbene or NO₃⁻) had no effect on the PPase activity. Vanadate and azide were also ineffective. H⁺-pumping PPase was inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N′-dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H⁺-PPase and the H⁺-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H⁺ pumping activity was strongly affected by Mg²⁺ and pyrophosphate (PPi) concentrations. At 5 millimolar Mg²⁺, H⁺ pumping showed a K_maPP for PPi of 15 micromolar. The rate of H⁺ pumping at 60 micromolar PPi was often equivalent to that at 1.5 millimolar ATP. The results suggest PPi hydrolysis could provide another source of a proton motive force used for solute transport and other energy-requiring processes across the tonoplast and other membranes with H⁺-PPase.     

Sodium-Stimulated NO(3) Uptake in Amaranthus tricolor L. Plants

Nitrate uptake of Na⁺ -deficient Amaranthus tricolor L. cv Tricolor seedlings from complete culture solution was stimulated by about 210% within 5 hours by application of 0.5 millimolar NaCl. From a Na⁺ -preloading experiment, intracellular Na⁺ was shown to be responsible for the stimulation of NO₃⁻ uptake. The results suggest a possible role of Na⁺ in NO₃⁻ uptake in C₄ plants.     

Electrogenic ATPase Activity on the Peribacteroid Membrane of Soybean (Glycine max L.) Root Nodules

Electrogenic ATPase activity on the peribacteroid membrane from soybean (Glycine max L. cv Bragg) root nodules is demonstrated. Membrane energization was monitored using suspensions of intact peribacteroid membrane-enclosed bacteroids (peribacteroid units; PBUs) and the fluorescent probe for membrane potential (ΔΨ), bis-(3-phenyl-5-oxoisoxazol-4yl) pentamethine oxonol. Generation of a positive ΔΨ across the peribacteroid membrane was dependent upon ATP, inhibited by N,N′-dicyclohexyl-carbodiimide and vanadate, but insensitive to N-ethylmaleimide, azide, cyanide, oligomycin, and ouabain. The results suggest the presence of a single, plasma membrane-like, electrogenic ATPase on the peribacteroid membrane. The protonophore, carbonyl-cyanide m-chlorophenyl hydrazone, completely dissipated the established membrane potential. The extent of reduction in the steady state membrane potential upon addition of ions was used to estimate the relative permeability of the peribacteroid membrane to anions. By this criterion, the relative rates of anion transport across the peribacteroid membrane were: NO₃⁻ > NO₂⁻ > Cl⁻ > acetate⁻ > malate⁻. The observation that 10 millimolar NO₃⁻ completely dissipated the membrane potential was of particular interest in view of the fact that NO₃⁻ inhibits symbiotic nitrogen fixation. The possible function of the ATPase in symbiotic nitrogen fixation is discussed.     

Evidence for cotransport of nitrate and protons in maize roots : I. Effects of nitrate on the membrane potential

The electrical response of nitrate-grown maize (Zea mays L.) roots to 0.1 millimolar nitrate was comprised of two sequential parts: a rapid and transient depolarization of the membrane potential, followed by a slower, net hyperpolarization to a value more negative than the original resting potential. The magnitude of the response was smaller in roots of seedlings grown in the absence of nitrate, but, within 3 hours of initial exposure to 0.1 millimolar nitrate, increased to that of nitrate-grown roots. Chloride elicited a separate electrical response with a pattern similar to that of the nitrate response. However, the results presented in this study strongly indicate that the electrical response to nitrate reflects the activity of a nitrate-inducible membrane transport system for nitrate which is distinct from that for chloride. Inhibitors of the plasmalemma H⁺-ATPase (vanadate, diethylstilbestrol) completely inhibited both parts of the electrical response to nitrate, as did alkaline external pH. The magnitude of the initial nitrate-dependent, membrane potential depolarization was independent of nitrate concentration, but the subsequent nitrate-dependent hyperpolarization showed saturable dependence with an apparent K_m of 0.05 millimolar. These results support a model for nitrate uptake in maize roots which includes a depolarizing NO₃⁻/H⁺ symport. The model proposes that the nitrate-dependent membrane potential hyperpolarization is due to the plasma membrane proton pump, which is secondarily stimulated by the operation of the NO₃⁻/H⁺ symport.     

NaCl Induces a Na/H Antiport in Tonoplast Vesicles from Barley Roots

Evidence was found for a Na⁺/H⁺ antiport in tonoplast vesicles isolated from barley (Hordeum vulgare L. cv California Mariout 72) roots. The activity of the antiport was observed only in membranes from roots that were grown in NaCl. Measurements of acridine orange fluorescence were used to estimate relative proton influx and efflux from the vesicles. Addition of MgATP to vesicles from a tonoplast-enriched fraction caused the formation of a pH gradient, interior acid, across the vesicle membranes. EDTA was added to inhibit the ATPase, by chelating Mg²⁺, and the pH gradient gradually dissipated. When 50 millimolar K⁺ or Na⁺ was added along with the EDTA to vesicles from control roots, the salts caused a slight increase in the rate of dissipation of the pH gradient, as did the addition of 50 millimolar K⁺ to vesicles from salt-grown roots. However, when 50 millimolar Na⁺ was added to vesicles from salt-grown roots it caused a 7-fold increase in the proton efflux. Inclusion of 20 millimolar K⁺ and 1 micromolar valinomycin in the assay buffer did not affect this rapid Na⁺/H⁺ exchange. The Na⁺/H⁺ exchange rate for vesicles from salt-grown roots showed saturation kinetics with respect to Na⁺ concentration, with an apparent K_m for Na⁺ of 9 millimolar. The rate of Na⁺/H⁺ exchange with 10 millimolar Na⁺ was inhibited 97% by 0.1 millimolar dodecyltriethylammonium.     

Inhibition of Nitrate Transport by Anti-Nitrate Reductase IgG Fragments and the Identification of Plasma Membrane Associated Nitrate Reductase in Roots of Barley Seedlings

Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO₃⁻ uptake by more than 90% but had no effect on NO₂⁻ uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO₃⁻ uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO₃⁻ uptake. The results present the possibility that NO₃⁻ uptake and NO₃⁻ reduction in the PM of barley roots may be related.     

Regulation of Vacuolar pH of Plant Cells: II. A P NMR Study of the Modifications of Vacuolar pH in Isolated Vacuoles Induced by Proton Pumping and Cation/H Exchanges

The vacuolar pH and the trans-tonoplast ΔpH modifications induced by the activity of the two proton pumps H⁺-ATPase and H⁺-PPase and by the proton exchanges catalyzed by the Na⁺/H⁺ and Ca²⁺/H⁺ antiports at the tonoplast of isolated intact vacuoles prepared from Catharanthus roseus cells enriched in inorganic phosphate (Y Mathieu et al 1988 Plant Physiol [in press]) were measured using the ³¹P NMR technique. The H⁺-ATPase induced an intravacuolar acidification as large as 0.8 pH unit, building a trans-tonoplast ΔpH up to 2.2 pH units. The hydrolysis of the phosphorylated substrate and the vacuolar acidification were monitored simultaneously to estimate kinetically the apparent stoichiometry between the vectorial proton pumping and the hydrolytic activity of the H⁺-ATPase. A ratio of H⁺ translocated/ATP hydrolyzed of 1.97 ± 0.06 (mean ± standard error) was calculated. Pyrophosphate-treated vacuoles were also acidified to a significant extent. The H⁺-PPase at 2 millimolar PPi displayed hydrolytic and vectorial activities comparable to those of the H⁺-ATPase, building a steady state ΔpH of 2.1 pH units. Vacuoles incubated in the presence of 10 millimolar Na⁺ were alkalinized by 0.4 to 0.8 pH unit. It has been shown by using ²³Na NMR that sodium uptake was coupled to the H⁺ efflux and occurred against rather large concentration gradients. For the first time, the activity of the Ca²⁺/H⁺ antiport has been measured on isolated intact vacuoles. Ca²⁺ uptake was strongly inhibited by NH₄Cl or gramicidin. Vacuoles incubated with 1 millimolar Ca²⁺ were alkalinized by about 0.6 pH unit and this H⁺ efflux was associated to a Ca²⁺ uptake as demonstrated by measuring the external Ca²⁺ concentration with a calcium specific electrode. Steady state accumulation ratios of Ca²⁺ as high as 100 were reached for steady state external concentrations about 200 micromolar. The rate of Ca²⁺ uptake appeared markedly amplified in intact vacuoles when compared to tonoplast vesicles but the antiport displayed a much lower affinity for calcium. The different behavior of intact vacuoles compared to vesicles appears mainly to be due to differences in the surface to volume ratio and in the rates of dissipation of the pH gradient. Despite its low affinity, the Ca²⁺/H⁺ antiport has a high potential capacity to regulate cytoplasmic concentration of calcium.     

Characterization of Anion Effects on the Nitrate-Sensitive ATP-Dependent Proton Pumping Activity of Soybean (Glycine max L.) Seedling Root Microsomes

The ATP-dependent proton-pumping activity of soybean (Glycine max L.) root microsomes is predominantly nitrate sensitive and presumably derived from the tonoplast. We used microsomes to characterize anion effects on proton pumping of the tonoplast vesicles using two distinctly different techniques.

Preincubation of the vesicles with nitrate caused inhibition of proton pumping and ATPase activity, with similar concentration dependence. Fluoride, which preferentially inhibits the plasma membrane ATPase, inhibited ATPase activity strongly at concentrations which did not affect proton pumping activity.

Addition of potassium salts, after a steady-state pH gradient is established in the absence of such salts, caused an increased pH gradient which was due to alleviation of Δ Ψ and subsequent increased influx of H⁺ into these vesicles. This anion-induced increase in the pH gradient could be used as a measure of the relative anion permeabilities, which were of the order Br⁻ = NO₃⁻ > Cl⁻ SO₄²⁻. Phosphate and fluoride caused no increase in the pH gradient. Since the concentration dependence of KCl- and KNO₃-induced quenching exhibited a saturable component, and since H⁺ uptake was increased by only certain anions, the data suggest that there may be a relatively specific anion channel associated with tonoplast-derived vesicles.