Tonoplast ATPase Research Articles

A Ca2+/H+ Antiport System Driven by the Proton Electrochemical Gradient of a Tonoplast H+-ATPase from Oat Roots

Two types of ATP-dependent calcium (Ca(2+)) transport systems were detected in sealed microsomal vesicles from oat roots. Approximately 80% of the total Ca(2+) uptake was associated with vesicles of 1.11 grams per cubic centimeter and was insensitive to vanadate or azide, but inhibited by NO(3) (-). 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 Mg(2+).Like the tonoplast H(+)-ATPase activity, vanadate-insensitive Ca(2+) 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 Ca(2+) transport system had an apparent K(m) for Mg-ATP of 0.24 millimolar similar to the tonoplast ATPase. The vanadate-insensitive Ca(2+) transport was abolished by compounds that eliminated a pH gradient and Ca(2+) 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 Ca(2+) accumulation into right-side-out tonoplast vesicles via a Ca(2+)/H(+) antiport. This transport system was saturable with respect to Ca(2+) (K(m) apparent = 14 micromolar). The Ca(2+)/H(+) antiport operated independently of the H(+)-ATPase since an artifically imposed pH gradient (acid inside) could also drive Ca(2+) accumulation. Ca(2+) transport by this system may be one major way in which vacuoles function in Ca(2+) homeostasis in the cytoplasm of plant cells.

The function of tonoplast ATPase in intact vacuoles of red beet is governed by direct and indirect ion effects

The pH gradient and the electric potential across the tonoplast in mechanically isolated beetroot vacuoles has been studied by following the uptake of [(14)C]methylamine and [(14)C]triphenyl-methylphosphoniumchloride. In response to Mg-ATP, the vacuolar interior is acidified by 0.8 units. This strong acidification is accompanied by a slight hyperpolarization of the membrane potential, which is probably caused by a proton diffusion potential. In preparations where only a small acidification (0.4 units) occurred, the membrane potential was depolarized by the addition of Mg-ATP. Different monovalent cations and anions were tested concerning their effect on the pH gradient and ATPase activity in proton-conducting tonoplasts. Chloride stimulation and NO 3 (-) inhibition were clearly present. The observed decline of the pH gradient upon the addition of Na(+) salts is probably caused by an Na(+)/H(+) antiport system.

Identification of 3-O-(4-benzoyl)benzoyladenosine 5‘-triphosphate- and N,N‘-dicyclohexylcarbodiimide-binding subunits of a higher plant H+-translocating tonoplast ATPase.

The polypeptide composition of the NO-3-sensitive H+-ATPase of vacuolar membrane (tonoplast) vesicles isolated from red beet (Beta vulgaris L.) storage root was investigated by affinity labeling with [alpha-32P]3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate [( alpha-32P]BzATP) and [14C]N,N'-dicyclohexylcarbodiimide [( 14C]DCCD). The photoactive affinity analog of ATP, BzATP, is a potent inhibitor of the tonoplast ATPase (apparent KI = 11 microM) and the photolysis of [alpha-32P]BzATP in the presence of native tonoplast yields one major 32P-labeled polypeptide of 57 kDa. Photoincorporation into the 57-kDa polypeptide shows saturation with respect to [alpha-32P]BzATP concentration and is blocked by ATP. [14C]DCCD, a hydrophobic carboxyl reagent and potent irreversible inhibitor of the tonoplast ATPase (k50 = 20 microM) labels a 16-kDa polypeptide in native tonoplast. The tonoplast ATPase is purified approximately 12-fold by Triton X-100 solubilization and Sepharose 4B chromatography. Partial purification results in the enrichment of two prominent polypeptides of 67 and 57 kDa. Solubilization, chromatography, and sodium dodecylsulfate-polyacrylamide gel electrophoresis of tonoplast labeled with [alpha-32P]BzATP or [14C]DCCD results in co-purification of the 57- and 16-kDa labeled polypeptides with ATPase activity. It is concluded that the tonoplast H+-ATPase is a multimer containing structurally distinct BzATP- and DCCD-binding subunits of 57 and 16 kDa, respectively. The data also suggest the association of a 67-kDA polypeptide with the ATPase.

Similarities and differences between the tonoplast-type and the mitochondrial H+-ATPases of oat roots.

The native tonoplast and the mitochondrial H+-ATPase from oat roots were compared to determine whether the two enzymes have similar mechanisms. H+ pumping in low-density microsomal vesicles reflected activity from the tonoplast-type ATPase, as ATPase activity and ATP-dependent H+ pumping (quinacrine fluorescence quenching) showed similar sensitivities to inhibition by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonate, nitrate, quercetin, or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. The tonoplast-type ATPase was stimulated by C1-,Br- greater than HCO3- whereas the mitochondrial ATPase was stimulated by HCO3- much greater than C1-,Br-. Both enzymes hydrolyzed ATP preferentially and were inhibited competitively by AMP or ADP. Apart from resistance to azide, the tonoplast-type ATPase was strikingly similar in its inhibitor sensitivities to the mitochondrial ATPase. The insensitivity to vanadate of both enzymes suggests the reaction mechanisms do not involve a covalent phosphoenzyme. Inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide and protection by ATP suggests tyrosine and cysteine residues are in the catalytic site of the tonoplast ATPase. The mitochondrial ATPase was 100 times more sensitive to N,N'-dicyclohexyl-carbodiimide inhibition than the tonoplast H+-ATPase. These results suggest the tonoplast and the mitochondrial H+-ATPases share common steps in their catalytic and vectorial reaction mechanisms, yet sufficient differences exist to indicate they are two distinct ATPases.

H+-ATPase Activity from Storage Tissue of Beta vulgaris

Two distinct membrane fractions containing H(+)-ATPase activity were prepared from red beet. One fraction contained a H(+)-ATPase activity that was inhibited by NO(3) (-) while the other contained a H(+)-ATPase inhibited by vanadate. We have previously proposed that these H(+)-ATPases are associated with tonoplast (NO(3) (-)-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 MgATP(2-), and Mg(2+) 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 Mg(2+)) 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 MgATP(2-) 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 MgATP(2-) 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 Mg(2+) could modulate ATPase activity at both the tonoplast and plasma membrane.

Partial Purification of a Tonoplast ATPase from Corn Coleoptiles

The tonoplast ATPase from corn coleoptile membranes was solubilized using a two-step procedure consisting of a pretreatment with 0.15% (w/v) deoxycholate to remove 60% of the protein, and 40 millimolar octyl-glucoside to solubilize the ATPase. During ultracentrifugation, the solublized ATPase entered a linear sucrose gradient faster than the majority of the protein, resulting in an 11-fold purification over the initial specific activity. The partially purified ATPase was almost completely inhibited by KNO(3) with an estimated K(i) of 10 millimolar. The specific activity of the KNO(3)-sensitive ATPase was increased 29-fold during purification. N,N'-Dicyclohexylcarbodiimide also completely inhibited the ATPase with half-maximal effects at a concentration of 4 micromolar. Neither vanadate nor azide inhibited enzyme activity. The purified ATPase was stimulated by Cl(-) and preferred Mg-ATP as substrate. Analysis of frations from the sucrose gradient by sodium dodecyl sulfate-polyacrylamide gel electrophoresis led to the identification of two major polypeptides at 72,000 and 62,000 daltons which were best correlated with ATPase activity. Several minor bands also appeared to copurify with enzyme activity, but were less consistent. Radiation inactivation experiments with intact membranes indicated that the functional molecular size of the tonoplast ATPase was nearly 400,000 daltons. This suggests that the ATPase is composed of several polypeptides, possibly including the 72,000- and 62,000-dalton proteins.

Preparation of Membrane Vesicles Enriched in ATP-Dependent Proton Transport from Suspension Cultures of Tomato Cells

Membranes enriched in ATP-dependent proton transport were prepared from suspension cultures of tomato cells (Lycopersicon esculentum Mill cv VF36). Suspension cultures were a source of large quantities of membranes from rapidly growing, undifferentiated cells. Proton transport activity was assayed as quench of acridine orange fluorescence. The activity of the proton translocating ATPase and of several other membrane enzymes was measured as a function of the cell culture cycle. The relative distribution of the enzymes between the 3,000, 10,000, and 100,000g pellets remained the same throughout the cell culture cycle, but yield of total activity and activity per gram fresh weight with time had a unique profile for each enzyme tested. Maximal yield of the proton translocating ATPase activity was obtained from cells in the middle logarithmic phase of growth, and from 50 to 90% of the activity was found in the 10,000g pellet. The proton translocating ATPase activity was separable from NADPH cytochrome c reductase and cytochrome c oxidase on a sucrose gradient. Proton transport activity had a broad pH optimum (7.0-8.0), was stimulated by KCl with a K(m) of 5 to 10 millimolar, stimulation being due to the anion, Cl(-), and not the cation, K(+), and was not inhibited by vanadate, but was inhibited by NO(3) (-). The activity is tentatively identified as the tonoplast ATPase.

Effect of magnesium and ATP on ATPase of sugarcane vacuoles

Kinetic analysis of the Mg(2+)-dependence of tonoplast ATPase from suspension-cultured cells of sugarcane showed that the enzyme activity increased with increasing magnesium concentrations till 1-3 mM and then decreased consideably for higher concentrations. This kinetic could be explained by the assumption that MgATP(2-) is the substrate of ATPase: MgATP(2-) concentration increases with increasing concentration of magnesium till, at high concentrations of magnesium, Mg2ATP is formed. No evidence for a direct role of Mg(2+) as activator or inhibitor was found. These data corroborate previous findings that MgATP(2-) is the sole substrate of the vacuolar ATPase of sugarcane (Thom and Komor 1984). High concentrations of ATP seemed to inhibit the ATPase. This result, however, could be traced back to interference of ATP with the Fiske-Subbarow method of phosphate determination. After adjustment of the test conditions, inhibition by ATP was no longer found. Reported data for ATPases of other plant materials, showing inhibition of enzyme activity with high magnesium or ATP concentrations, might be explicable in a similar way.

H+-ATPase Activity from Storage Tissue of Beta vulgaris

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 NO(3) (-)-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 NO(3) (-)-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 NO(3) (-) 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 KNO(3), stimulated by anions (Cl(-) > Br(-) > acetate > HCO(3) (-) > SO(4) (2-)), 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.

Role of the ATPase of sugar‐cane vacuoles in energization of the tonoplast

Vacuoles of sugar-cane suspension cells contained a tonoplast-bound ATPase which was exclusively located on the cytoplasmic side of the vacuole. Vanadate and diethylstilbestrol had little effect on the vacuolar ATPase. ATP was the optimum substrate for the tonoplast ATPase, but there was also evidence for tonoplast-bound GDP-hydrolyzing and GTP-hydrolyzing enzymes which can interfere with the ATPase assay. Other phosphate anhydrides and esters were not hydrolyzed. The addition of MgATP polarized the tonoplast from about 0 mV to an interior-positive value of about +20 mV; MgADP and MgGTP had much less effect; MgGDP and ATP (in the absence of magnesium) had no effect on the membrane potential. The polarization of the tonoplast was insensitive to valinomycin, nigericin, and inhibitors of plasmalemma ATPase, but was strongly reduced by the uncoupler carbonylcyanide m-chlorophenylhydrazone. These data are interpreted as evidence for the action of tonoplast-bound ATPase as a pump which translocates protons into the vacuoles. The activity of the ATPase was highly specific for MgATP2-; the other important ionic states of ATP:ATP4-, HATP3-, MgHATP-, and Mg2ATP neither stimulated nor inhibited. The same was true for Mg2+. Since the protons were not brought to the catalytic site by protonation of the substrate, the tonoplast-ATPase may pick up the proton for translocation from the cytoplasm. The saturation kinetics for MgATP2- hydrolysis were biphasic, the higher affinity ATPase with Km value of 0.7 mM seems to be the physiologically relevant activity.

Sensitivity of Tonoplast-Bound Adenosine-Triphosphatase from Hevea to Inhibitors

The tonoplast-bound H(+)-translocating ATPase from Hevea latex was found to be insensitive to vanadate, diethylstilbestrol, and octylguanidine, which are specific inhibitors of the plasma membrane ATPase. The inhibitors of the mitochondrial ATPase, oligomycin and azide, and also rotenone and antimycin A, were all without effect. In contrast, trimethyltin chloride strongly inhibited the activity of Hevea tonoplast ATPase.Among the different carbodiimides tested, which strongly inhibit the Hevea tonoplast ATPase, N,N'-dicyclohexylcarbodiimide was the most inhibitory. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline was also an efficient inhibitor.This unique inhibitor sensitivity of the Hevea tonoplast H(+)-translocating ATPase suggests that this enzyme differs in its mode of operation from all other known H(+)-translocating ATPases.

Characterization and solubilization of nucleotide-specific, Mg 2+-ATPase and Mg 2+-pyrophosphatase of tonoplast

Nucleotide-specific, Mg 2+-dependent ATPase and Mg 2+-dependent pyrophosphatase were recovered with purified tonoplast obtained from isolated Tulipa petal vacuoles. Relative Mg 2+-dependent hydrolysis of ATP, GTP and pyrophosphate, the only substrates hydrolyzed to a substantial degree, was 1.0, 0.3, and 0.6, respectively. Tonoplast ATPase required Mg 2+, and essentially no Mg 2+-dependent or Mg 2+-independent p- nitrophenylphosphatase (which was associated with intact vacuoles) occurred with the membrane. Tonoplast ATPase was stimulated 10 to 30% by KCl, but was little effected by other cations (other than NH 4 +) or anions. No activity was observed with CaATP as substrate. The enzyme was cold stable and was inhibited by DCCD and Dio-9, but not by oligomycin. Its pH optimum was 7.0 and its specific activity was about 50 μmol P i/mg protein per h at 37°C. Properties of membrane-bound and Polidocanol (polyoxyethylene ether, 9 lauryl ether) solubilized enzyme were similar. Reduced activity of solubilized enzyme was partially restored with phospholipids. Tonoplast ATPase appears to be an integral membrane component which requires phospholipids for maximal activity. Tonoplast Mg 2+-pyrophosphatase had a pH optimum of ≥ 8.5, was stimulated 2.5-fold by 50 mM KCl, and was largely lost upon detergent treatment. Properties of tonoplast ATPase observed are consistent with the characteristics of proton transport exhibited by isolated, intact Tulipa vacuoles (Wagner, G.J. and Lin, W. (1982) Biochim. Biophys. Acta 689, 261–266). These observations suggest that tonoplast ATPase functions in proton transport.

Evidence for an electrogenic adenosine-triphosphatase in Hevea tonoplast vesicles

The function of the Mg-dependent ATPase of Hevea tonoplast in active proton transport was investigated by using a purified tonoplast fraction containing tightly sealed vesicles. In the used experimental conditions, the uptake of [(14)C]triphenylmethyl-phosphonium ion ([(14)C]TPMP(+)) and [(3)H]tetraphenyl-phosphonium ion ([(3)H]TPP(+)) by the vesicles indicated a transmembrane potential difference, negative inside. In parallel, the uptake of [(14)C]methylamine into the vesicles monitored a transmembrane pH gradient, interior acid. The addition of 5 mM Mg-ATP markedly depolarized the membrane and increased the magnitude of trnasmembrane pH gradient. These ATP-driven events were substrate specific for Mg-ATP. They were strongly inhibited by ATPase inhibitors such as N, N'-dicyclohexylcar-bodiimide. They were completely eliminated by proton conductors such as carbonylcyanide-p-trifluoromethoxy-phenylhydrazone and 5-chloro-3-tert-butyl-2'-chloro-4-nitro-salicylanilide. They depended on the pH of the medium, the maximum being reached at about pH 7.0. These data provide in vitro evidence that the Mg-ATPase localized at tonoplast level is an electrogenic pump. They are consistent with the hypothesis that an electrogenic H(+) pump is catalyzed by the tonoplast ATPase of higher plants.

Characterization of a Proton-Translocating ATPase in Microsomal Vesicles from Corn Roots

Sealed microsomal vesicles were prepared from corn (Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000g microsomal fraction onto a 10% dextran T-70 cushion. The Mg(2+)-ATPase activity of the sealed vesicles was stimulated by Cl(-) and NH(4) (+) and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The ionophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 6.5. The ATPase was inhibited by NO(3) (-), was insensitive to K(+), and was not inhibited by 100 micromolar vanadate or by 1 millimolar azide.Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg(2+), was stimulated by Cl(-), inhibited by NO(3) (-), was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATP-dependent fluorescence quench both required a divalent cation (Mg(2+) >/= Mn(2+) > Co(2+)) and were inhibited by high concentrations of Ca(2+). The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the proton-translocating ATPase differed from those of the mitochondrial F(1)F(0)-ATPase and from those of the K(+)-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase.

Characterization of Nigericin-Stimulated ATPase from Sealed Microsomal Vesicles of Tobacco Callus

To understand the function and membrane origin of ionophore-stimulated ATPases, the activity of nigericin-stimulated ATPase was characterized from a low-density microsomal fraction containing sealed vesicles of autonomous tobacco (Nicotiana tabacum Linnaeous cv. Wisconsin no. 38) callus. The properties of KCl-stimulated, Mg-requiring ATPases (KCl-Mg,ATPase) were similar in the absence or presence of nigericin. Nigericin (or gramicidin) stimulation of a KCl-Mg,ATPase activity was optimum at pH 6.5 to 7.0. The enzyme was inhibited completely by N,N'-dicyclohexylcarbodiimide (10 mum), tributyltin (5 mum), and partially by vanadate (200 mum), but it was insensitive to fusicoccin and mitochondrial ATPase inhibitors, such as azide (1 mm) and oligomycin (5 mug/ml). The ATPase was more sensitive to anions than cations. Cations stimulated ATPase activity with a selectivity sequence of NH(4) (+) > K(+), Rb(+), Cs(+), Na(+), Li(+) > Tris(+). Anions stimulated Mg, ATPase activity with a decreasing sequence of Cl(-) = acetate > SO(4) (2-) > benzene sulfonate > NO(3) (-). The anion stimulation was caused partly by dissipation of the electrical potential (interior positive) by permeant anions and partly by a specific ionic effect. Plant membranes had at least two classes of nigericin-stimulated ATPases: one sensitive and one insensitive to vanadate. Many of the properties of the nigericin-sensitive, salt-stimulated Mg,ATPase were similar to a vanadate-sensitive plasma membrane ATPase of plant tissues, yet other properties (anion stimulation and vanadate insensitivity) resembled those of a tonoplast ATPase. These results support the idea that nigericin-stimulated ATPases are mainly electrogenic H(+) pumps originated in part from the plasma membrane and in part from other nonmitochondrial membranes, such as the tonoplast.

An active proton pump of intact vacuoles isolated from Tulipa petals

Anthocyanin pigments within Tulipa petal vacuoles provide the means for real-time spectrophotometric monitoring of vacuolar sap pH and for studying ATP-dependent proton transport in isolated, intact vacuoles. Spectra of petal extracts were used to select empirically those wavelengths giving an approximately linear variation in anthocyanin absorbance with pH over a pH range of interest. A sensitive single-beam spectrophotometer with vertical optics was used to minitor absorbance changes of intact, settled vacuoles. Substrates and inhibitors of vacuolar ATPase (Lin, W., Wagner, G.J., Siegelman, H.W. and Hind, Q. (1977) Biochim. Biophys. Acta 465, 110–117) were added to probe proton transport. Acidification of the vacuole sap occurred following addition of MgATP, but not CaATP. Proton accumulation was inhibited by 10 μM Dio 9, an inhibitor of tonoplast ATPase in vitro, and the proton gradient established by addition of MgATP was dissipated after addition of 10 μM CCCP. No pumping response was observed with intact protoplasts. Potential differences across the tonoplast were directly measured by impaling vacuoles with glass microelectrodes. Potential differences of 10–20 mV (inside positive) were recorded when vacuoles were suspended in 0.7 M mannitol/10 mM Hepes buffer (adjusted to pH 8.0 with KOH), and 0.5 mM dithiothreitol. Addition of MgATP increased the potential difference by 2–5 mV.

Further evidence for the proton pumping work of tonoplast ATPase from Hevea latex vacuome

An ATP-dependent acidification of internal medium of Hevea tonoplast vesicles could be monitored by 9-amino-6-chloro-2-methoxyacridine. The reported data indicate a lack of effect of typical plasmalemma ATPase inhibitors (DES, vanadate) on the proton pumping activity of Hevea tonoplast ATPase. A role of this tonoplast ATPase is evoked in the regulation of cytoplasm pH.