Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue

Intact vacuoles were isolated from petals of Hippeastrum and Tulipa (Wagner G.J. and Siegelman, H.W. (1975) Science 190, (1298–1299). The ATPase activity of fresh vacuole suspensions was found to be 2–3 times that of protoplasts from the same tissue. 70–80% of the ATPase activity of intact vacuoles was recovered in tonoplast preparations. The antibiotic Dio-9 at 6 μg/10⁶ vacuoles or protoplasts causes 40% inhibition. However, only the protoplast ATPase is sensitive to oligomycin. N,N′-dicyclohexylcarbodiimide (DCCD) slightly stimulates ATPase activity in both vacuole and protoplast suspensions, whereas ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDAC) strongly inhibits.Spectrophotometric studies show that in the petal the vacuolar contents have a pH of 4.0 for Tulipa and 4.3 for Hippeastrum, whereas the intact isolated vacuole has an internal pH of 7.0 (in pH 8.0 buffer) for Tulipa and about 7.3 for Hippeastrum. Internal ion concentrations of 150, 46, 30, 30 and 6 mM were found for K⁺, Na⁺, Mg²⁺, Cl^?, and Ca²⁺ respectively, which are about the same as those in protoplasts.     

Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue.

Intact vacuoles were isolated from petals of Hippeastrum and Tulipa (Wagner G.J. and Siegelman, H.W. (1975) Science 190, 1298--1299). The ATPase activity of fresh vacuole suspensions was found to be 2--3 times that of protoplasts from the same tissue. 70--80% of the ATPase activity of intact vacuoles was recovered in tonoplast preparations. The antibiotic Dio-9 at 6mug/10(6) vacuoles or protoplasts causes 40% inhibition. However, only the protoplast ATPase is sensitive to oligomycin. N,N'-dicyclohexylcarbodiimide (DCCD) slightly stimulates ATPase activity in both vacuole and protoplast suspensions, whereas ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDAC) strongly inhibits. Spectrophotometric studies show that in the petal the vacuolar contents have a pH of 4.0 for Tuplipa and 4.3 for Hippeastrum, whereas the intact isolated vacuole has an internal pH of 7.0 (in pH 8.0 buffer) for (Tulipa and about 7.3 for Hippeastrum. Internal ion concentrations of 150, 46, 30, 30 and 6 mM were found for K+, Na+, Mg2+, Cl-, and Ca2+ respectively, which are about the same as those in protoplasts.     

Two Proton Pumps Operate in Parallel Across the Tonoplast of Vacuoles Isolated from Suspension Cells of Chenopodium rubrum L.*

The kinetics of vacuolar acidification upon addition of ATP and/or pyrophosphate (PPi) has been assayed on single immobilized vacuoles by computer-aided microfluorimetry of 9-aminoacridine, and by acridine orange absorption photometry on vacuole suspensions isolated from green suspension cells of Chenopodium rubrum L. Two proton pumps at the tonoplast, an ATPase and a pyrophosphatase (PPase), operate in parallel to acidify the vacuole with different contributions adding up to a transtonoplast Δ pH of 2.6 pH units at external pH 7.2. The saturable components of proton pumping reach half maximal velocity with 0.32 ± 0.06 mM ATP and 23 ± 2.5 μM PPi, respectively. At saturating substrate concentrations, ATPase and PPase hydrolyse ATP and PPi, respectively, at a ratio of 2.3. The same ratio holds for the corresponding proton fluxes maintaining a given steady-state vacuolar pH. We conclude that both pumps operate at the same stoichiometry.     

Electrogenic proton translocation by the ATPase of sugarcane vacuoles

Existence of a proton-translocating ATPase on the tonoplast of higher plants has been further confirmed by use of two experimental systems: (a) intact isolated vacuoles from sugarcane cells and (b) vesicles prepared from the same source. Addition of MgATP to vacuoles polarized the tonoplast by 40 millivolts to a value of +20 millivolts, but a large preexisting pH gradient across the membrane restricted the pH change to 0.2 unit. In vesicle preparations, the tonoplast was polarized to +66 millivolts by the addition of MgATP and the intravesicular space was acidified by 1 pH unit to pH 5.5. Proton translocation equilibrium is controlled by the protonmotive potential difference, maximal at 125 millivolts for sugarcane cells. Energization of the tonoplast occurred at physiological concentrations of MgATP. Specificity of MgATP for proton translocation was indicated by a much smaller effect of MgADP and MgGDP on the electrochemical gradient, although these substrates were also hydrolyzed by tonoplast preparation.     

The properties of arginine transport in vacuolar membrane vesicles of Neurospora crassa

We have measured the uptake of arginine into vacuolar membrane vesicles from Neurospora crassa. Arginine transport was found to be dependent on ATP hydrolysis, Mg2+, time, and vesicle protein with transported arginine remaining unmodified after entry into the vesicles. The Mg2+ concentration required for optimal arginine transport varied with the ATP concentration so that maximal transport occurred when the MgATP2- concentration was at a maximum and the concentrations of free ATP and Mg2+ were at a minimum. Arginine transport exhibited Michaelis-Menten kinetics when the arginine concentration was varied (Km = 0.4 mM). In contrast, arginine transport did not follow Michaelis-Menten kinetics when the MgATP2-concentration was varied (S0.5 = 0.12 mM). There was no inhibition of arginine transport when glutamine, ornithine, or lysine were included in the assay mixture. In contrast, arginine transport was inhibited 43% when D-arginine was present at a concentration 16-fold higher than that of L-arginine. Measurements of the internal vesicle volume established that arginine is concentrated 14-fold relative to the external concentration. Arginine transport was inhibited by dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl-hydrazone, and potassium nitrate (an inhibitor of vacuolar ATPase activity). Inhibitors of the plasma membrane or mitochondrial ATPase such as sodium vanadate or sodium azide did not affect arginine transport activity. In addition, arginine transport had a nucleoside triphosphate specificity similar to that of the vacuolar ATPase. These results suggest that arginine transport is dependent on vacuolar ATPase activity and an intact proton channel and proton gradient.     

Bafilomycin Inhibits Vacuolar pH Regulation in a Fresh Water Charophyte,Chora corallina

Bafilomycin A₁, known as an inhibitor of vacuolar type H⁺-ATPase, was used to study involvement of the vacuolar ATP-dependent H⁺-pump in the vacuolar pH regulation in a fresh water charophyte, Chara corallina. When bafilomycin A₁ (100 nM) was externally given to intact cells, the vacuolar pH (about 5) was not affected. Internodal cells were then pretreated with 100 nM bafilomycin for 1 ? 2 h and the vacuolar sap was replaced with a weakly buffered solution of pH 7.4. The readjustment of the modified vacuolar pH in bafilomycin-treated cells was significantly retarded compared with that in untreated cells. Next, bafilomycin A₁ was directly introduced into the vacuole by vacuolar perfusion with the artificial cell sap of pH 7.4. At 100 nM bafilomycin A₁, the decrease in the vacuolar pH was significantly inhibited. When cell sap was replaced with the artificial cell sap containing no buffer (pH 5.2 ? 5.5), the vacuolar pH increased in the presence of vacuolar bafilomycin, suggesting that the PP₁- dependent H⁺ pumping alone was not sufficient for the pH regulation of Chara vacuoles. Intracellular bafilomycin A₁ had no effect on the plasma membrane potential of tonoplast-free cells, which is evidence that it does not affect the electrogenic H⁺-pump in the plasma membrane. Bafilomycin A₁ inhibited the ATP-dependent H⁺ transport of tonoplast vesicles but not the PP₁-dependent H⁺ transport. The ATPase activity of tonoplast vesicles was also inhibited by bafilomycin A₁.     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

The structure of sialyl-glycopeptides of the O-glycosidic type, isolated from sialidosis (mucolipidosis I) urine

Plants performing crassulacean acid metabolism show a large nocturnal accumulation of malic acid in the vacuole of the photosynthetic cells. It has been postulated that an H+-translocating ATPase energizes the transport of malic acid across the tonoplast into the vacuole. In the present work we have characterized the ATPase activity associated with vacuoles of the crassulacean-acid-metabolism plant Kalancho? daigremontiana and compare it with other phosphohydrolases. Vacuoles were isolated by polybase-induced lysis of mesophyll-cell protoplasts. The vacuoles had a high activity of unspecific acid phosphatase (pH optimum 5.3). The acid phosphatase was strongly inhibited by ammonium molybdate (with 50% inhibition at about 0.5 mmol m-3), but was not completely inhibited even at much higher ammonium-molybdate concentrations. In contrast, the vacuolar ATPase activity, assayed in the presence of 100 mmol m-3 ammonium molybdate, had a pH optimum of 8.0. ATP was the preferred substrate, but GTP, ITP and ADP were hydrolyzed at appreciable rates. The mean ATPase activity at pH 8.0 was 14.5 nmol h-1 (10(3) vacuoles)-1, an average 13% of which was attributable to residual acid-phosphatase activity. Inorganic-pyrophosphatase activity could not be demonstrated unambiguously. The vacuolar ATPase activity was Mg2+-dependent, had an apparent Km for MgATP2- of 0.31 mol m-3, and was 32% stimulated by 50 mol m-3 KCl. Of the inhibitors tested, oligomycin slightly inhibited the vacuolar ATPase activity and diethylstilbestrol and NO-3 were both markedly inhibitory. Dicyclohexylcarbodiimide and tributyltin were also strongly inhibitory. Tributyltin caused a 50% inhibition at about 0.3 mmol m-3. This is taken as evidence that the vacuolar ATPase might function as an H+-translocating ATPase. It is shown that the measured activity of the vacuolar ATPase would be of the right order to account for the observed rates of nocturnal malic-acid accumulation in K. daigremontiana.     

The lysolipid sphingosine modulates pyrophosphatase activity in tonoplast vesicles and isolated vacuoles from a heterotrophic cell suspension culture of Chenopodium rubrum

The proton pumping activity of the tonoplast (vacuolar membrane) H⁺-ATPase and H⁺-pyrophosphatase (H⁺-PPase) has been studied on a tonoplast-enriched microsomal fraction and on intact vacuoles isolated from a heterotrophic cell suspension culture of Chenopodium rubrum L. in the presence of the lysosphingolipids D-sphingosine, psychosine (galactosylsphingosine) and lysosulfatide (sulfogalactosyl-sphingosine). Sphingosine strongly stimulates (K_a= 0.16 μ M ) the PPase activity, assayed both as ΔpH formation across the tonoplast vesicle membrane, and as reversible clamp current measured by the whole-vacuolar mode of the patch-clamp technique. Psychosine showed a minor, and lysosulfatide no stimulatory effect. No effect upon the ATPase activity has been observed. No sphingosine-induced change could be observed in the affinity of the PPase for its substrate (apparent K_m= 10 μ M MgPPi). We tentatively conclude that sphingosine, which is known as a potent inhibitor of the protein kinase C in animal cells, may be a regulator of the plant vacuolar PPase.     

Characteristics of Sucrose Transport and Sucrose-Induced H+ Transport on the Tonoplast of Red Beet (Beta vulgaris L.) Storage Tissue

Sucrose-induced changes of the energization state of the red beet root (Beta vulgaris L. ssp. conditiva) vacuolar membrane were observed with the fluorescent dyes 6-chloro-9-{[4-(diethylamino)- 1-methylbutyl]-amino}-2-methoxyacridine dihydrochloride, as a pH monitor, and 9-amino-6-chloro-2-methoxyacridine (ACMA). Consequently, transient acidification of the surrounding suspension medium could be measured with a pH electrode. This signal was specific for Suc and was not seen for sorbitol, mannitol, or maltose. Sucrose-induced medium acidification was sensitive to the same inhibitors that were efficient in inhibiting sucrose transport, including the monoclonal antibodies TNP56-12 and C50-5-3. It was seen with vacuoles and vesicles energized with MgATP before sucrose was added but also with vacuoles not artificially energized previously. Using bafilomycin A1 for the inhibition of the vacuolar ATPase of vacuoles previously energized by MgATP, apparent Km values for H+ export from the vacuoles to the medium could be calculated taking into account the passive proton leak. Apparent Km values for H+ export determined from data obtained with pH measurements in the medium and with ACMA corresponded to those obtained previously for sucrose uptake. Comparing sucrose uptake rates with corresponding H+ export rates at the respective sucrose concentrations and at Km, a stoichiometry of approximately one proton per transported sucrose was estimated.     

The effect of rishitin on potato tonoplast vesicle and vacuole proton transport

Rishitin, a known potato phytoalexin, was tested for its effects on proton transport. Like the pterocarpan phytoalexins, glyceollin and phaseollin, rishitin was found to inhibit proton transport. At 100 μM rishitin, proton transport in potato tonoplast vesicles was inhibited by >95%. This inhibition appears to be due to an increase in proton conductance and not to inhibition of the tonoplast ATPase. Potato vacuoles were also shown to have increased proton leakage in the presence of rishitin.     

Effects of ATP analogs on the proton pumping by the vacuolar H+-ATPase from maize roots

Understanding the regulatory properties of the activities of the V-type adenosine triphosphatase (ATPase) on tonoplast membranes is important in determining the mechanisms by which this enzyme controls cytoplasmic and vacuolar pH. The possible existence of a regulatory site for adenine nucleotides was examined by comparing the effects of ADP, adenylylimidodiphosphate (AMP-PNP) and 3'- o -(4-benzoyl) benzoyladenine 5'-triphosphate (BzATP) to those of the 2',3'-dialdehyde derivative of AMP (oAMP) and ATP by using highly purified tonoplast vesicles from maize ( Zea mays L. cv. FRB 73) roots. The addition of either AMP-PNP or BzATP reversibly inhibited the initial rate of proton transport catalyzed by the H⁺-ATPase in a concentration-dependent manner. Less than 20 μ M AMP-PNP or 50 μ M BzATP was sufficient to inhibit half the initial rate of proton transport in the presence of 2 m M ATP and an excess of Mg. Both analogs increased the K_m for ATP and reduced the maximum enzyme velocity. The presence of ADP also inhibited proton transport. The characteristics of ADP-induced inhibition were similar to those of BzATP and AMP-PNP. The addition of the periodated derivative of AMP (oAMP) irreversibly inhibited the ATPase in a concentration and time-dependent manner similar to that reported previously (Chow et al. 1992, Plant Physiology 98: 44–52). Irreversible inhibition by oAMP reduced the maximum velocity of the tonoplast ATPase and was prevented by the addition of ATP. The presence of ADP, AMP-PNP or BzATP had no effect on irreversible inhibition by oAMP. The effects of ADP, AMP-PNP and BzATP on the kinetics of ATP utilization and the lack of protection against inhibition by oAMP argue in favor of at least two types of nucleotide binding sites on the V-type ATPase from maize root tonoplast membranes.     

Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue

Modification of our previous procedure for the isolation of microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue allowed the recovery of sealed membrane vesicles displaying proton transport activity sensitive to both nitrate and orthovanadate. In the absence of a high salt concentration in the homogenization medium, contributions of nitrate-sensitive (tonoplast) and vanadate-sensitive (plasma membrane) proton transport were roughly equal. The addition of 0.25 M KCl to the homogenization medium increased the relative amount of nitrate-inhibited proton transport activity while the addition of 0.25 M KI resulted in proton pumping vesicles displaying inhibition by vanadate but stimulation by nitrate. These effects appeared to result from selective sealing of either plasma membrane or tonoplast membrane vesicles during homogenization in the presence of the two salts. Following centrifugation on linear sucrose gradients it was shown that the nitrate-sensitive, proton-transporting vesicles banded at low density and comigrated with nitrate-sensitive ATPase activity while the vanadate-sensitive, proton-transporting vesicles banded at a much higher density and comigrated with vanadate-sensitive ATPase. The properties of the vanadate-sensitive proton pumping vesicles were further characterized in microsomal membrane fractions produced by homogenization in the presence of 0.25 M KI and centrifugation on discontinuous sucrose density gradients. Proton transport was substrate specific for ATP, displayed a sharp pH optimum at 6.5, and was insensitive to azide but inhibited by N'-N-dicyclohexylcarbodiimide, diethylstilbestrol, and fluoride. The Km of proton transport for Mg:ATP was 0.67 mM and the K0.5 for vanadate inhibition was at about 50 microM. These properties are identical to those displayed by the plasma membrane ATPase and confirm a plasma membrane origin for the vesicles.     

Concanamycin 4-B: A Potent Inhibitor of Vacuolar pH Regulation in Chara Cells

Concanamycin 4-B, a macrolide antibiotic with an 18-membered lactone ring, is known as a specific inhibitor of the vacuolar type of H⁺-ATPase, as is bafilomycin A₁. The drug was tested for its effect on regulation of the vacuolar pH (pH_v) of internodal cells of a fresh water characean alga, Chara corallina, under normal conditions and under salt stress. The pH_v was measured either on isolated vacuolar sap with a conventional pH electrode or directly by inserting a pH-sensitive glass microelectrode into the vacuole. Proton-pumping into tonoplast vesicles was almost completely inhibited by concanamycin 4-B at 1 nM. Concanamycin 4-B at 1 μM significantly increased pH_v while bafilomycin A₁ was ineffective when applied at 1 μM. Concanamycin 4-B did not affect pH_v when applied at 0.1 μM and increasing the concentration to 10 μM did not amplify the degree of alkalization. Concanamycin 4-B also inhibited pH_v regulation under NaCl stress. When Chara cells were treated with 100 mM NaCl, pH_v promptly increased and then recovered to the original level. The reacidification was completely inhibited by concanamycin 4-B (1 μM), suggesting that the reacidification was achieved by the H⁺-ATPase of the tonoplast.     

Effects of W-7, W-5, Verapamil and Diltiazem on vacuolar proton transport. Comparison of vacuolar H+-ATPase and H+-PPase from roots of Zea mays

The vacuolar membrane of plant cells is characterized by two proton pumps: the vacuolar H⁺-ATPase (V-ATPase; EC 3.6.1.3) and the vacuolar H⁺-PPase (V-PPase; EC 3.6.1.1). Recently, Du Pont and Morrissey reported that Ca²⁺ stimulates hydrolytic activity of purified V-ATPase (Arch. Biochim. Biophys., 1992. 294: 341–346). Since this effect may be due to degradation during purification further investigation of Ca²⁺ regulation of native V-ATPase was done. However, native tonoplast membranes contain a Ca²⁺/H⁺ antiport activity, which interferes with effects of calcium ions on proton transport activity of vacuolar ATPase. Therefore, the effects of anti-calmodulin drugs (W-7, W-5, calmidazolium), and calcium channel antagonists (Verapamil, Diltiazem) on proton transport activities of the vacuolar-type H⁺-ATPase and H⁺-PPase in tonoplast enriched membrane vesicle preparations from roots of Zea mays L. were studied. The concentrations for half maximal inhibition of vacuolar H⁺-ATPase (H⁺-PPase) were: 71 (191) μM W-7, 470 (> 800) μM W-5, 26 (24) μM calmidazolium (= compound R 24571). 398 (700) μM Verapamil, and 500 (1 330) μM Diltiazem. Estimation of Hill coefficients (n_H) for the inhibition by Verapamil showed a further difference between the two vacuolar proton pumps (H⁺-ATPase, n_H= 2.02; H⁺-PPase, n_n= 0.96). The data indicate that the vacuolar H⁺-ATPase itself is affected by these chemicals. It is suggested that some biological activities of W-7, W-5, Verapamil, and Diltiazem are due to their effects on proton translocation by the vacuolar-type H⁺-ATPase.     

Elicitor-activated phospholipase A(2) generates lysophosphatidylcholines that mobilize the vacuolar H(+) pool for pH signaling via the activation of Na(+)-dependent proton fluxes

The elicitation of phytoalexin biosynthesis in cultured cells of California poppy involves a shift of cytoplasmic pH via the transient efflux of vacuolar protons. Intracellular effectors of vacuolar proton transport were identified by a novel in situ approach based on the selective permeabilization of the plasma membrane for molecules of < or = 10 kD. Subsequent fluorescence imaging of the vacuolar pH correctly reported experimental changes of activity of the tonoplast proton transporters. Lysophosphatidylcholine (LPC) caused a transient increase of the vacuolar pH by increasing the Na(+) sensitivity of a Na(+)-dependent proton efflux that was inhibited by amiloride. In intact cells, yeast elicitor activated phospholipase A(2), as demonstrated by the formation of LPC from fluorescent substrate analogs, and caused a transient increase of endogenous LPC, as determined by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. It is suggested that LPC generated by phospholipase A(2) at the plasma membrane transduces the elicitor-triggered signal into the activation of a tonoplast H(+)/Na(+) antiporter.     

Transport of 1-aminocyclopropane-1-carboxylic acid into isolated maize mesophyll vacuoles

Intracellular transport of the ethylene precursor, I-aminocyclopropane-1-carboxylic acid (ACC) can change the ACC concentration in cell compartments and impact ethylene biosynthesis. Transport of ACC into isolated maize ( Zea mays L.) mesophyll vacuoles was studied by silicon layer flotation filtering. The transport of ACC across the tonoplast was stimulated 2. 4- to 8. 1-fold by 5 m M MgATP, showed saturation kinetics with an apparent K_m for ACC of 20 μ M , and was optimal at 25°C. Transport of ACC was sensitive to the pH of the medium, falling as external pH rose. Effectors known to inhibit proton-translocating ATPases (N, N-dicyclohexylcarbodiimide) and to collapse the electrical (thiocyanate, valinomycin) and chemical (carbonylcyanide m -chlorophenylhydrazone, gramicidin) potential gradients for protons across the tonoplast all reduced ACC transport. The nonhydrolyzable MgATP analog. Mg adenylyl-imidodiphosphate, stimulated ACC transport as effectively as MgATP. Other nucleotides (MgADP, MgCTP, MgUTP, MgGTP) and MgPP_i had little or no effect. These results suggest that ACC uptake into isolated maize mesophyll vacuoles is carrier mediated, is dependent upon an electrochemical potential gradient for protons and is specifically regulated, but not necessarily energized, by MgATP     

Voltage sensitivity of H+/Ca2+ antiport in higher plant tonoplast suggests a role in vacuolar calcium accumulation

The electrogenicity of H+/Ca2+ exchange in vacuolar membrane (tonoplast) vesicles from Beta was studied to elucidate the role of this transport system in vacuolar Ca2+ accumulation. To overcome the inherently high proton permeability of tonoplast vesicles, the pH difference established by the primary H(+)-ATPase was titrated to a uniform value by variation of the concentration either of ATP or of a permanent anion (Cl-). This enabled manipulation of membrane potential independently of the transmembrane pH difference, with a higher inside-positive membrane potential produced at lower Cl- concentrations. The rate and the extent of uncoupler-sensitive Ca2+ uptake are both stimulated about 2-fold in conditions of more positive membrane potential, suggesting that the transport system translocates positive charge outward during Ca2+ uptake. A minimum integral H+:Ca2+ stoichiometry of 3 results in a driving force for Ca2+ accumulation in the vacuole amounting to -140 mV in typical physiological conditions. It is concluded that the antiporter is thermodynamically competent to account for Ca2+ accumulation in plant vacuoles and that its reversal in vivo is unlikely.     

Vacuolar transport of the glutathione conjugate of trans-cinnamic acid

Red beet (Beta vulgaris L.) tonoplast membrane vesicles and [14C]trans-cinnamic acid-glutatione were used to study the vacuolar transport of phynylpropanoid-glutathione conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [14C]trans-cinnamic acid-glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-conjugated [14C]trans-cinnamic acid. Uptake of the conjugate in the presence of MgATP was not dependent on a trans-tonoblast H+-electrochemical gradient, because uptake was not affected by the addition of NH4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 microM; 14% inhibition). Uptake of the conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4-dinitrophenyl) glutathione (500 microM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated Km and Vmax values for uptake were 142 microM amd 5.95 nmol mg(-1) min(-1), respectively. Based on these results, phenylpropanoid-glutation conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the glutathione S-conjugate pump(s) located in the tonoplast membrane.     

Differential energization of the tonoplast in suspension cells and seedlings from Picea abies

 Vacuolar ATPase (EC 3.6.1.3) and PPase (EC 3.6.1.1) were studied in suspension cells and seedlings from spruce [Picea abies (L.) Karst. Proton transport activity and uncoupler (1 μM nigericin) stimulated substrate hydrolysis were measured in tonoplast enriched membrane vesicles. In suspension cells the vacuolar PPase exhibited 1.8-fold activity of the ATPase. In roots and needles from 12-week-old spruce seedlings the vacuolar PPase was inactive, whereas the ATPase was active. Therefore, we investigated whether the preparation of spruce tonoplast vesicles from roots and needles inactivates the vacuolar PPase but not the ATPase. For this purpose, maize (Zea mays L.) tonoplast membranes exhibiting vacuolar PPase as well as ATPase activity were used as a probe and added to the homogenization medium prior to the preparation of spruce vesicles. The preparation of spruce vesicles was more inhibitory to the vacuolar ATPase than to the PPase. The comparison of vacuolar PPases from spruce suspension cells and maize roots revealed similar enzymatic properties. After isopycnic centrifugation on continuous sucrose gradients the vacuolar PPase from spruce suspension cells co-purified with the vacuolar ATPase. Together, these data show: (1) vacuolar PPases from spruce suspension cells and maize roots are similar, (2) the preparation of tonoplast vesicles from spruce roots and needles does not inactivate the vacuolar PPase, (3) tonoplasts of suspension cultured cells and seedlings from spruce are differentially energized by the vacuolar pyrophosphatase that may indicate a difference in pyrophosphate metabolism between embryogenic and differentiated spruce cells, and (4) tonoplast vesicles from spruce seedlings may allow investigations of the effect of pyrophosphate on the vacuolar ATPase in the absence of vacuolar PPase activity. Received: 2 July 1998 / Accepted: 14 September 1998