Transport Properties of the Tomato Fruit Tonoplast : I. Identification and Characterization of an Anion-Sensitive H-ATPase

An anion-sensitive H⁺-translocating ATPase was identified in membrane vesicles isolated from mature green tomato (Lycopersicon esculentum) fruit. The H⁺-ATPase was associated with a low density membrane population having a peak density of 1.11 grams per cubic centimeter, and its activity was inhibited by NO₃⁻, N,N′-dicyclohexylcarbodiimide and diethylstilbestrol but not by vanadate, azide, molybdate, or oligomycin. This H⁺-ATPase has an unusual pH dependence indicating both a slightly acidic and a near neutral peak of activity. Chloride was found to be a potent stimulator of ATPase activity. The K_m for the H⁺-ATPase was approximately 0.8 millimolar ATP. The characteristics of this H⁺-ATPase are very similar to those described for a number of plant cell tonoplast H⁺-ATPases suggesting that the activity identified in tomato fruit membranes is tonoplast-associated. This report demonstrates the feasibility of isolating tonoplast vesicles from acidic fruit tissues for studies of transport activities associated with fruit development and maturation.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products

Two distinct membrane fractions containing H⁺-ATPase activity were prepared from red beet. One fraction contained a H⁺-ATPase activity that was inhibited by NO₃⁻ while the other contained a H⁺-ATPase inhibited by vanadate. We have previously proposed that these H⁺-ATPases are associated with tonoplast (NO₃⁻-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP²⁻, and Mg²⁺ concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg²⁺) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.

Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP²⁻ in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.

Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP²⁻ complex.

The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H⁺-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg²⁺ could modulate ATPase activity at both the tonoplast and plasma membrane.

     

Determination of H/ATP Stoichiometry for the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue

The H⁺/ATP stoichiometry was determined for the plasma membrane H⁺-ATPase from red beet (Beta vulgaris L., var Detroit Dark Red) storage tissue associated with native vesicles. The determination of H⁺/ATP stoichiometry utilized a kinetic approach where rates of H⁺ influx, estimated by three different methods, were compared to rates of ATP hydrolysis measured by the coupled enzyme assay under identical conditions. These methods for estimating H⁺ influx were based upon either determining the initial rate of alkalinization of the external medium from pH 6.13, measuring the rate of vesicle H⁺ leakage from a steadystate pH gradient after stopping the H⁺-ATPase or utilizing a mathematical model which describes the net transport of H⁺ at any given point in time. When the rate of H⁺ influx estimated by each of these methods was compared to the rate of ATP hydrolysis, a H⁺/ATP stoichiometry of about 1 was observed. In consideration of the maximum free energy available from ATP hydrolysis (ΔG_atp), this value for H⁺/ATP stoichiometry is sufficient to account for the magnitude of the proton electrochemical gradient observed across the plasma membrane in vivo.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: I. Identification and Characterization of an Anion-Sensitive H-ATPase

Microsomal membranes isolated from red beet (Beta vulgaris L.) storage tissue were found to contain high levels of ionophore-stimulated ATPase activity. The distribution of this ATPase activity on a continuous sucrose gradient showed a low density peak (1.09 grams per cubic centimeter) that was stimulated over 400% by gramicidin and coincided with a peak of NO₃⁻-sensitive ATPase activity. At higher densities (1.16-1.18 grams per cubic centimeter) a shoulder of gramicidin-stimulated ATPase that coincided with a peak of vanadate-sensitive ATPase was apparent. A discontinuous sucrose gradient of 16/26/34/40% sucrose (w/w) was effective in routinely separating the NO₃⁻-sensitive ATPase (16/26% interface) from the vanadate-sensitive ATPase (34/40% interface). Both membrane fractions were shown to catalyze ATP-dependent H⁺ transport, with the transport process showing the same differential sensitivity to NO₃⁻ and vanadate as the ATPase activity.

Characterization of the lower density ATPase (16/26% interface) indicated that it was highly stimulated by gramicidin, inhibited by KNO₃, stimulated by anions (Cl⁻ > Br⁻ > acetate > HCO₃⁻ > SO₄²⁻), and largely insensitive to monovalent cations. These characteristics are very similar to those reported for tonoplast ATPase activity and a tonoplast origin for the low density membrane vesicles was supported by comparison with isolated red beet vacuoles. The membranes isolated from the vacuole preparation were found to possess an ATPase with characteristics identical to those of the low density membrane vesicles, and were shown to have a peak density of 1.09 grams per cubic centimeter. Furthermore, following osmotic lysis the vacuolar membranes apparently resealed and ATP-dependent H⁺ transport could be demonstrated in these vacuole-derived membrane vesicles. This report, thus, strongly supports a tonoplast origin for the low density, anion-sensitive H⁺-ATPase and further indicates the presence of a higher density, vanadate-sensitive, H⁺-ATPase in the red beet microsomal membrane fraction, which is presumably of plasma membrane origin.

     

Enhanced H Transport Capacity and ATP Hydrolysis Activity of the Tonoplast H-ATPase after NaCl Adaptation

Tonoplast enriched membrane vesicle fractions were isolated from unadapted and NaCl (428 millimolar) adapted tobacco cells (Nicotiana tabacum L. var Wisconsin 38). Polypeptides from the tonoplast enriched vesicle fractions were separated by SDS-PAGE and analyzed by Western blots using polyclonal antibodies to the 70 kilodalton subunit of the red beet tonoplast H⁺-ATPase. These antibodies cross-reacted exclusively to a tobacco polypeptide of an apparent molecular weight of 69 kilodaltons. The antibodies inhibited ATP-dependent, NO₃⁻ sensitive H⁺ transport into vesicles in tonoplast enriched membrane fractions from both unadapted and NaCl adapted cells. The relative H⁺ transport capacity per unit of 69 kilodalton subunit of the tonoplast ATPase of vesicles from NaCl adapted cells was fourfold greater than that observed for vesicles from unadapted cells. The increase in specific H⁺ transport capacity after adaptation was also observed for ATP hydrolysis.     

Mechanism of Stimulation and Inhibition of Tonoplast H-ATPase of Beta vulgaris by Chloride and Nitrate

The H⁺-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl⁻ and NO₃⁻. N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the K_m of the enzyme for ATP but caused relatively little alteration of the V_max. Nitrate increased K_m only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl⁻. Other monovalent anions that specifically increased the K_m of the ATPase for ATP were, in order of increasing K_i, SCN⁻, ClO₄⁻, and ClO₃⁻. Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO₃⁻, was a competitive inhibitor of the ATPase but ADP and NO₃⁻ did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO₃⁻ does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg·ADP and Mg·ATP protected the enzyme from inactivation by NEM but Mg·ADP was the more potent of the two. Chloride and NO₃⁻ exerted little or no effect on the protective actions of Mg·ADP and Mg·ATP suggesting that neither Cl⁻ nor NO₃⁻ are involved in substrate binding.     

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.     

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.     

Characterization of a NO(3)-Sensitive H-ATPase from Corn Roots

When assayed in the presence of azide, NO₃⁻ was shown to be a specific inhibitor of a proton-translocating ATPase present in corn (Zea mays L. cv WF9 × M017) root microsomal membranes. The distribution of the NO₃⁻-sensitive ATPase on sucrose gradients and its general characteristics are similar to those previously reported for the anion-stimulated H⁺-ATPase of corn roots believed to be of tonoplast origin. An ATPase inhibited by 20 μm vanadate and insensitive to molybdate was also identified in corn root microsomal membranes which could be largely separated from the NO₃⁻-sensitive ATPase on sucrose gradients and is believed to be of plasma membrane origin. Inasmuch as both ATPase most likely catalyze the efflux of H⁺ from the cytoplasm, our objective was to characterize and compare the properties of both ATPases under identical experimental conditions. The vanadate-sensitive ATPase was stimulated by cations (K⁺ > NH₄⁺ > Rb⁺ > Cs⁺ > Li⁺ > Na⁺ > choline⁺) whereas the NO₃⁻-sensitive ATPase was stimulated by anions (Cl⁻ > Br⁻ > C₂H₃O₂⁻ > SO₄²⁻ > I⁻ > HCO₃⁻ > SCN⁻). Both ATPases required divalent cations. However, the order of preference for the NO₃⁻-sensitive ATPase (Mn²⁺ > Mg²⁺ > Co²⁺ > Ca²⁺ > Zn²⁺) differed from that of the vanadate-sensitive ATPase (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺). The vanadate-sensitive ATPase required higher concentrations of Mg:ATP for full activity than did the NO₃⁻-sensitive ATPase. The kinetics for Mg:ATP were complex for the vanadate-sensitive ATPase, indicating positive cooperativity, but were simple for the NO₃⁻-sensitive ATPase. Both ATPases exhibited similar temperature and pH optima (pH 6.5). The NO₃⁻-sensitive ATPase was stimulated by gramicidin and was associated with NO₃⁻-inhibitable H⁺ transport measured as quenching of quinacrine fluorescence. It was insensitive to molybdate, azide, and vanadate, but exhibited slight sensitivity to ethyl-3-(3-dimethylaminopropyl carbodiimide) and mersalyl. Overall, these results indicate several properties which distinguish these two ATPases and suggest that under defined conditions NO₃⁻-sensitive ATPase activity may be used as a quantitative marker for those membranes identified tentatively as tonoplast in mixed or nonpurified membrane fractions. We feel that NO₃⁻ sensitivity is a better criterion by which to identify this ATPase than either Cl⁻ stimulation or H⁺ transport because it is less ambiguous. It is also useful in identifying the enzyme following solubilization.     

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.     

Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes

The effects of NO₃⁻ and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H⁺-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO₃ when assayed at 24°C or above and was tentatively identified as the tonoplast H⁺-ATPase. A smaller peak of H⁺-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO₃ and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H⁺-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO₃ inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO₃. The time course for fluorescence quench indicated that addition of ATP in the presence of KNO₃ caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO₃ was explained by the temperature-dependent delay of the inhibition by KNO₃. The plasma membrane H⁺-ATPase maintained a pH gradient in the presence of KNO₃ for up to 30 minutes at 24°C.     

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.     

ADP Is a Competitive Inhibitor of ATP-Dependent H Transport in Microsomal Membranes from Zea mays L. Coleoptiles

An anion-sensitive ATP-dependent H⁺ transport in microsomal membranes from Zea mays L. coleoptiles was partially characterized using the pH gradient-dependent decrease of unprotonated neutral red. The following criteria strongly suggest a tonoplast origin of the H⁺ transport observed: strict dependence on Cl⁻; inhibition by SO₄²⁻ and NO₃⁻; insensitivity against vanadate, molybdate, and azide; reversible inhibition by CaCl₂ (H⁺/Ca²⁺ antiport); inhibition by diethylstilbestrol. The substrate kinetics revealed simple Michaelis Menten kinetics for ATP in the presence of 1 millimolar MgCl₂ with a K_m value of 0.56 millimolar (0.38 millimolar for MgATP). AMP and c-AMP did not influence H⁺ transport significantly. However, ADP was a potent competitive inhibitor with a K_i value of 0.18 millimolar. The same inhibition type was found for membranes prepared from primary roots by the same procedure.     

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.     

A Ca/H Antiport System Driven by the Proton Electrochemical Gradient of a Tonoplast H-ATPase from Oat Roots

Two types of ATP-dependent calcium (Ca²⁺) transport systems were detected in sealed microsomal vesicles from oat roots. Approximately 80% of the total Ca²⁺ uptake was associated with vesicles of 1.11 grams per cubic centimeter and was insensitive to vanadate or azide, but inhibited by NO₃⁻. The remaining 20% was vanadate-sensitive and mostly associated with the endoplasmic reticulum, as the transport activity comigrated with an endoplasmic reticulum marker (antimycin A-insensitive NADH cytochrome c reductase), which was shifted from 1.11 to 1.20 grams per cubic centimeter by Mg²⁺.

Like the tonoplast H⁺-ATPase activity, vanadate-insensitive Ca²⁺ accumulation was stimulated by 20 millimolar Cl⁻ and inhibited by 10 micromolar 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid or 50 micromolar N,N′-dicyclohexylcarbodiimide. This Ca²⁺ transport system had an apparent K_m for Mg-ATP of 0.24 millimolar similar to the tonoplast ATPase. The vanadate-insensitive Ca²⁺ transport was abolished by compounds that eliminated a pH gradient and Ca²⁺ dissipated a pH gradient (acid inside) generated by the tonoplast-type H⁺-ATPase. These results provide compelling evidence that a pH gradient generated by the H⁺-ATPase drives Ca²⁺ accumulation into right-side-out tonoplast vesicles via a Ca²⁺/H⁺ antiport. This transport system was saturable with respect to Ca²⁺ (K_m apparent = 14 micromolar). The Ca²⁺/H⁺ antiport operated independently of the H⁺-ATPase since an artifically imposed pH gradient (acid inside) could also drive Ca²⁺ accumulation. Ca²⁺ transport by this system may be one major way in which vacuoles function in Ca²⁺ homeostasis in the cytoplasm of plant cells.

     

Dissociation and Reassembly of the Vacuolar H-ATPase Complex from Oat Roots

Conditions for the dissociation and reassembly of the multi-subunit vacuolar proton-translocating ATPase (H⁺-ATPase) from oat roots (Avena sativa var Lang) were investigated. The peripheral sector of the vacuolar H⁺-ATPase is dissociated from the membrane integral sector by chaotropic anions. Membranes treated with 0.5 molar KI lost 90% of membrane-bound ATP hydrolytic activity; however, in the presence of Mg²⁺ and ATP, only 0.1 molar KI was required for complete inactivation of ATPase and H⁺-pumping activities. A high-affinity binding site for MgATP (dissociation constant = 34 micromolar) was involved in this destabilization. The relative loss of ATPase activity induced by KI, KNO₃, or KCl was accompanied by a corresponding increase in the peripheral subunits in the supernatant, including the nucleotide-binding polypeptides of 70 and 60 kilodaltons. The order of effectiveness of the various ions in reducing ATPase activity was: KSCN > KI > KNO₃ > KBr > K-acetate > K₂SO₄ > KCl. The specificity of nucleotides (ATP > GTP > ITP) in dissociating the ATPase is consistent with the participation of a catalytic site in destabilizing the enzyme complex. Following KI-induced dissociation of the H⁺-ATPase, the removal of KI and MgATP by dialysis resulted in restoration of activity. During dialysis for 24 hours, ATP hydrolysis activity increased to about 50% of the control. Hydrolysis of ATP was coupled to H⁺ pumping as seen from the recovery of H⁺ transport following 6 hours of dialysis. Loss of the 70 and 60 kilodalton subunits from the supernatant as probed by monoclonal antibodies further confirmed that the H⁺-ATPase complex had reassembled during dialysis. These data demonstrate that removal of KI and MgATP resulted in reassociation of the peripheral sector with the membrane integral sector of the vacuolar H⁺-ATPase to form a functional H⁺ pump. The ability to dissociate and reassociate in vitro may have implications for the regulation, biosynthesis, and assembly of the vacuolar H⁺-ATPase in vivo.     

Chilling-Induced Inactivation and Its Recovery of Tonoplast H-ATPase in Mung Bean Cell Suspension Cultures

The processes involved in adaptation to cold temperature were examined by growing suspension cultured cells of mung bean (Vigna radiata [L.] Wilczek) at 2°C for various periods of time and assaying the activities of various membrane-bound enzymes in vitro. The tonoplast H⁺-ATPase activity and the ATP-proton transport extracted from cells incubated at 2°C declined rapidly and reached a minimum level after 10 hours. The inactivation was reversible within 24 hours of chilling. The recovery of the cold-inactivated H⁺-ATPase was found to proceed in two steps, a faster recovery of ATP hydrolysis activity and a slower recovery of the proton transport. The recovery was markedly inhibited by the presence of azide, but not affected by 0.578 millimolar cycloheximide. This suggested the involvement of an energy process that had no requirement for de novo synthesis of protein. The cold-induced inactivation of the H⁺-ATPase may be due to a structural alteration of the enzyme. The slower recovery of proton transport relative to ATP hydrolysis during warming suggests that the protogenic domains in the enzyme may be affected differently by chilling.     

Substrate kinetics of the tonoplast h-translocating inorganic pyrophosphatase and its activation by free mg

To clarify the kinetic characteristics and ionic requirements of the tonoplast H⁺-translocating inorganic pyrophosphatase (H⁺-PPiase), PPi hydrolysis and PPi-dependent H⁺ transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie. The tonoplast H⁺-PPiase showed an absolute requirement for a monovalent cation and exhibited hyperbolic kinetics with respect to cation concentration. H⁺-PPiase activity was maximal in the presence of K⁺ (K₅₀ approximately 3 millimolar), with PPi-dependent H⁺ transport being more selective for K⁺ than PPi hydrolysis. When assayed in the presence of 50 millimolar KCl at fixed PPi concentrations, H⁺-PPiase activity showed sigmoidal kinetics with respect to total Mg concentration, reflecting a requirement for a Mg/PPi complex as substrate and free Mg²⁺ for activation. At saturating concentrations of free Mg²⁺, H⁺-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi²⁻ but not Mg₂PPi, demonstrating that MgPPi²⁻ was the true substrate of the enzyme. The apparent K_m (MgPPi²⁻) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H⁺ transport (7 micromolar). Free Mg²⁺ was shown to be an allosteric activator of the H⁺-PPiase, with Hill coefficients of 2.5 for PPi hydrolysis and 2.7 for PPi-dependent H⁺ transport. Half-maximal H⁺-PPiase activity occurred at a free Mg²⁺ concentration of 1.1 millimolar, which lies within the range of accepted values for cytosolic Mg²⁺. In contrast, cytosolic concentrations of K⁺ and MgPPi²⁻ appear to be saturating for H⁺-PPiase activity. We propose that one function of the H⁺-PPiase may be to act as an ancillary enzyme that maintains the proton-motive force across the vacuolar membrane when the activity of the tonoplast H⁺-ATPase is restricted by substrate availability. As ATP levels decline in the cytosol, free Mg²⁺ would be released from the MgATP²⁻ complex, thereby activating the tonoplast H⁺-PPiase.     

Evidence for Cotransport of Nitrate and Protons in Maize Roots : II. Measurement of NO(3) and H Fluxes with Ion-Selective Microelectrodes

We report here on an investigation of net nitrate and proton fluxes in root cells of maize (Zea mays L.) seedlings grown without (noninduced) and with (induced) 0.1 millimolar nitrate. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, WJ Lucas [1987] Plant Physiol 84: 1177-1184) was utilized to quantify net ionic fluxes from the measurement of electrochemical potential gradients for NO₃⁻ and H⁺ within the unstirred layer at the root surface. The nitrate-inducibility, pH dependence, and concentration dependence of net NO₃⁻ uptake correlated quite closely with the electrical response of maize roots to nitrate under the same experimental conditions (as described in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289). Additionally, it was found that potential inhibitors of the plasmalemma H⁺-ATPase (vandate, diethylstilbestrol), which were shown to abolish the electrical response to NO₃⁻ (in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289), dramatically inhibited NO₃⁻ absorption. These results strongly indicate that the NO₃⁻ electrical response is due to the operation of a NO₃⁻ transport system in the plasmalemma of maize root cells. Furthermore, the results from the H⁺-ATPase inhibitor studies indicate that the NO₃⁻ transport system is linked to the H⁺-ATPase, presumably as a NO₃⁻/H⁺ symport. This is further supported by the pH response of the NO₃⁻ transport system (inhibition at alkaline pH values) and the change in net H⁺ flux from a moderate efflux in the absence of NO₃⁻, to zero net H⁺ flux after exposing the maize root to exogenous nitrate. Although these results can be explained by other interpretations, the simplest model that fits both the electrical responses and the NO₃⁻/H⁺ flux data is a NO₃⁻/H⁺ symport with a NO₃⁻:H⁺ flux stoichiometry >1, whose operation results in the stimulation of the H⁺-ATPase due to the influx of protons through the cotransport system.     

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.