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.

     

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.     

Proton transport in isolated vacuoles from corn coleoptiles

Vacuoles were isolated from corn coleoptile protoplasts and ATP-dependent proton transport was measured by quinacrine fluorescence quenching or by the uptake of [¹⁴C]methylamine. Intact vacuoles were judged to be free of a surrounding plasma membrane based on fluorescent staining with fluoroscein-diacetate. Essentially all of the detectable ATP-stimulated methylamine uptake and α-mannosidase activities present in intact protoplasts were recovered in isolated vacuoles. In contrast, the activities of marker enzymes for plasma membranes, Golgi, endoplasmic reticulum, and mitochondria were reduced to 5 to 17% in vacuolar preparations. The characteristics of proton pumping by isolated vacuoles were compared to those of light microsomal membranes possibly derived from the tonoplast. ATP-dependent proton pumping by both isolated vacuoles and light microsomal vesicles was stimulated by Cl⁻, and inhibited by NO₃⁻, carbonyl cyanide-m-chlorophenylhydrazone, N,N′-dicyclohexylcarbodiimide, N-ethylmaleimide, 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid, diethylstilbestrol, and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but not by vanadate. Both activities also showed substrate specificity for Mg-ATP. Finally, proton transport activities of vacuolar and microsomal fractions exhibited similar profiles after flotation in linear dextran gradients. We conclude that the microsomal proton pump previously characterized in corn coleoptiles (Mettler et al. 1982 Plant Physiol 70: 1738-1742) is derived from the tonoplast.     

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.     

An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite,Plasmodium falciparum

The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and ³⁶Cl⁻ flux measurements was used to investigate the transport of Cl⁻ and the Cl⁻-dependent transport of “H⁺-equivalents” in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl⁻, resulting in an outward [Cl⁻] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H⁺-equivalents). This was reversed on restoration of extracellular Cl⁻. The flux of H⁺-equivalents was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. ³⁶Cl⁻ influx measurements revealed the presence of a Cl⁻ uptake mechanism with characteristics similar to those of the Cl⁻-dependent H⁺-equivalent flux. The intracellular concentration of Cl⁻ in the parasite was estimated to be ∼48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl⁻ together with H⁺-equivalents, resulting in an intracellular Cl⁻ concentration well above that which would occur if Cl⁻ ions were distributed passively in accordance with the parasite''s large, inwardly negative membrane potential.     

Calcium transport in tonoplast and endoplasmic reticulum vesicles isolated from cultured carrot cells

Two active calcium (Ca²⁺) transport systems have been identified and partially characterized in membrane vesicles isolated from cultured carrot cells (Daucus carota Danvers). Both transport systems required MgATP for activity and were enhanced by 10 millimolar oxalate. Ca²⁺ transport in membrane vesicles derived from isolated vacuoles equilibrated at 1.10 grams per cubic centimeter and comigrated with Cl⁻-stimulated, NO₃⁻-inhibited ATPase activity on sucrose density gradients. Ca²⁺ transport in this system was insensitive to vanadate, but was inhibited by nitrate, carbonyl cyanide-m-chlorophenylhydrazone (CCCP), N,N′-dicyclohexylcarbodiimide (DCCD), and 4,4-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS). The K_m for MgATP and Ca²⁺ were 0.1 mm and 21 micromolar, respectively. The predominant Ca²⁺ transport system detectable in microsomal membrane preparations equilibrated at a density of 1.13 grams per cubic centimeter and comigrated with the endoplasmic reticulum (ER) marker, antimycin A-insensitive NADH-dependent cytochrome c reductase. Ca²⁺ transport activity and the ER marker also shifted in parallel in ER shifting experiments. This transport system was inhibited by vanadate (I₅₀ = 12 micromolar) and was insensitive to nitrate, CCCP, DCCD, and DIDS. Transport exhibited cooperative MgATP dependent kinetics. Ca²⁺ dependent kinetics were complex with an apparent K_m ranging from 0.7 to 2 micromolar. We conclude that the vacuolar-derived system is a Ca²⁺/H⁺ antiport located on the tonoplast and that the microsomal transport system is a Ca,Mg-ATPase enriched on the ER. These two Ca²⁺ transport systems are proposed to restore and maintain cytoplasmic Ca²⁺ homeostasis under changing cellular and environmental conditions.     

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.     

Ion transport in isolated protoplasts from tobacco suspension cells: I. General characteristics

An investigation was conducted into the feasibility of using enzymically isolated protoplasts from suspension-cultured cells of Nicotiana glutinosa L. to study ion transport. Transport of K⁺ (⁸⁶Rb), ³⁶Cl⁻, H₂³²PO₄⁻ and ⁴⁵Ca²⁺ from 1 millimolar salt solutions was determined after separation of intact protoplasts from nonabsorbed tracers by centrifugation through a Ficoll step gradient. Influx of K⁺, Cl⁻, and H₂PO₄⁻ measured over a 30-minute period was reduced (up to 99%) by respiratory inhibitors such as 5 micrograms per milliliter oligomycin, 0.1 millimolar dinitrophenol, 0.1 millimolar cyanide, or N₂ gas. In contrast, Ca²⁺ influx was not tightly coupled to respiratory energy production. The influx of K⁺ was highest between pH 6.5 and 7.5 whereas the influx of H₂PO₄⁻ and Cl⁻ was greatest between pH 4.5 and 5.5. Influx of K⁺ and Cl⁻ was maximal at 35 and 45 C, respectively, and was almost completely inhibited below 10 C. Fusicoccin (0.01 millimolar) stimulated K⁺ influx by more than 200% but had no effect on the influx of either Cl⁻ or H₂PO₄⁻. Apparent H⁺ efflux, as measured by decrease in solution pH, was enhanced by K⁺, stimulated further by 0.01 millimolar fusicoccin, and inhibited by 0.1 millimolar dinitrophenol or 5 micrograms per milliliter oligomycin. The measured ionic fluxes into protoplasts were similar to those obtained with intact cultured cells. The results indicate that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast, and that it is feasible to use isolated protoplasts for studies on ion transport.     

Solubilization and reconstitution of the oat root vacuolar h/ca exchanger

Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H⁺/Ca²⁺ antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [ΔpH] dependent). The first step toward purification and identification of the H⁺/Ca²⁺ antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H⁺/Ca²⁺ antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A ΔpH (acid inside) was generated in the proteoliposomes with an NH₄Cl gradient (NH₄⁺_in » NH₄⁺_out) as determined by methylamine uptake. Fundamental properties of ΔpH dependent calcium uptake such as the K_m for calcium (~15 micromolar) and the sensitivity to inhibitors such as N,N′-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H⁺/Ca²⁺ antiporter has been reconstituted in active form.     

Membrane transport in isolated vesicles from sugarbeet taproot : I. Isolation and characterization of energy-dependent, h-transporting vesicles

Sealed membrane vesicles were isolated from homogenates of sugarbeet (Beta vulgaris L.) taproot by a combination of differential centrifugation, extraction with KI, and dextran gradient centrifugation. Relative to the KI-extracted microsomes, the content of plasma membranes, mitochondrial membranes, and Golgi membranes was much reduced in the final vesicle fraction. A component of ATPase activity that was inhibited by nitrate co-enriched with the capacity of the vesicles to form a steady state pH gradient during the purification procedure. This suggests that the nitrate-sensitive ATPase may be involved in driving H⁺-transport, and this is consistent with the observation that H⁺-transport, in the final vesicle fraction was inhibited by nitrate. Proton transport in the sugarbeet vesicles was substrate specific for ATP, insensitive to sodium vanadate and oligomycin but was inhibited by diethylstilbestrol and N,N′-dicyclohexylcarbodiimide. The formation of a pH gradient in the vesicles was enhanced by halide ions in the sequence I⁻ > Br⁻ > Cl⁻ while F⁻ was inhibitory. These stimulatory effects occur from both a direct stimulation of the ATPase by anions and a reduction in the vesicle membrane potential. In the presence of Cl⁻, alkali cations reduce the pH gradient relative to that observed with bis-tris-propane, possibly by H⁺/alkali cation exchange. Based upon the properties of the H⁺-transporting vesicles, it is proposed that they are most likely derived from the tonoplast so that this vesicle preparation would represent a convenient system for studying the mechanism of transport at this membrane boundary.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

Plasmalemma Chloride Transport in Chara corallina: Inhibition by 4,4'-Diisothiocyano-2,2'-Disulfonic Acid Stilbene

Chloride transport, presumably via a Cl⁻-2H⁺ co-transport system, was investigated in Chara corallina. At pH 6.5, the control influx (3.1 picomoles per centimeter² per second) was stimulated 4-fold by an 18-hour Cl⁻ starvation. The stimulated influx was inhibited to 4.7 picomoles per centimeter² per second after a 60-minute pre-exposure to 0.5 millimolar 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS). This compares with a nonsignificant inhibition of the control under similar conditions. At 2 millimolar DIDS, both stimulated and control influx were inhibited to values of 1.1 and 2.2 picomoles per centimeter² per second, respectively; in all cases, DIDS inhibition was reversible. Over the pH range 4.8 to 8.5, the control and DIDS-inhibited influx showed only slight pH sensitivity; in contrast, the stimulated flux was strongly pH dependent (pH 6.5 optimum). Inasmuch as changes in pH alter membrane potential, N-ethylmaleimide was used to depolarize the membrane; this had no effect on Cl⁻ influx. A transient depolarization of the membrane (about 20 millivolts) was observed on restoration of Cl⁻ to starved cells. The membrane also depolarized transiently when starved cells were exposed to 0.5 millimolar DIDS, but the depolarization associated with Cl⁻ restoration was inhibited by a 40-minute pretreatment with DIDS. Exposure of control cells to DIDS caused only a small hyperpolarization (about 7 millivolts). DIDS may have blocked Cl⁻ influx by inhibiting the putative plasmalemma H⁺-translocating ATPase. Histochemical studies on intact cells revealed no observable effect of DIDS on plasmalemma ATPase activity. However, DIDS application after fixation resulted in complete inhibition of ATPase activity.

The differential sensitivity of the stimulated and control flux to inhibition by DIDS may reflect an alteration of transport upon stimulation, but could also result from differences in pretreatment. The stimulated cells were pretreated with DIDS in the absence of Cl⁻, in contrast to the presence of Cl⁻ during pretreatment of controls. The differential effect could result from competition between Cl⁻ and DIDS for a common binding site. Our histochemical ATPase results indicate that Cl⁻ transport and membrane ATPase are separate systems, and the latter is only inhibited by DIDS from the inside of the cell.

     

Pyrophosphate-driven proton transport by microsomal membranes of corn coleoptiles

Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on isopycnic sucrose density gradients. Two peaks of ATP-driven H⁺-transport activity, corresponding to the previously characterized tonoplast (1.07 grams per cubic centimeter) and Golgi (1.13 grams per cubic centimeter) fractions (Chanson and Taiz, Plant Physiol 1985 78: 232-240) were localized. Coincident with these were two peaks of inorganic pyrophosphate (PPi)-driven H⁺-transport. At saturating (3 millimolar) concentrations of Mg²⁺:ATP, the rate of proton transport was further enhanced by the addition of 3 millimolar PPi, and the stimulation was additive, i.e. equal to the sum of the two added separately. The specific PPi analog, imidodiphosphate, antagonized PPi-driven H⁺-transport, but had no effect on ATP-driven transport. Moreover, PPi-dependent proton transport in both tonoplast-enriched and Golgi-enriched fractions was strongly promoted by 50 millimolar KNO₃, unlike the ATP-dependent H⁺-pumps of the same membranes. Taken together, the results indicate that PPi-driven proton transport is mediated by specific membrane-bound H⁺-translocating pyrophosphatases. Both potassium and a permanent anion (NO₃⁻ > Cl⁻), were required for maximum activity. The PPi-driven proton pumps were totally inhibited by N,N′-dicyclohexylcarbodiimide, but were insensitive to 100 millimolar vanadate. The PPi concentration in coleoptile extracts was determined using an NADH oxidation assay system coupled to purified pyrophosphate:fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90). The total pyrophosphate content of corn coleoptiles was 20 nanomoles/gram fresh weight. Assuming a cytoplasmic location, the calculated PPi concentration is sufficient to drive proton transport at 20% of the maximum rate measured in vitro for the tonoplast-enriched fraction, and 10% of the maximum rate for the Golgi-enriched fraction.     

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.     

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.     

Inhibition of anion transport in corn root protoplasts

The effects of several amino-reactive disulfonic stilbene derivatives and N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate on Cl⁻, SO₄²⁻, and inorganic phosphate (Pi) uptake in protoplasts isolated from corn root tissue were studied. 4-Acetamido-4′-isothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diamino-2,2′-stilbenedisulfonic acid, and NAP-taurine inhibited Cl⁻ and SO₄²⁻ but not Pi and K⁺ uptake in corn root protoplasts; whereas mersalyl inhibited Pi but not Cl⁻ or SO₄²⁻ uptake. The rate of uptake of all anions decreased with increasing external pH. In addition, these reagents markedly inhibited plasmalemma ATPase activity isolated from corn root tissue. Excised root segments were less sensitive to Cl⁻ and SO₄²⁻ transport inhibitors.     

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

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

Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells: II. Selectivity and Kinetics

Protoplasts were enzymically isolated from suspension cultured cells of Nicotiana glutinosa L. and aspects of transport selectivity and kinetics were studied. In the presence of Ca²⁺, transport was selective for K⁺ (⁸⁶Rb) over Na⁺. ³⁶Cl⁻ transport was inhibited by Br⁻ or I⁻ but not by H₂PO₄⁻. The kinetic data for short term (30 minutes) K⁺ influx over the range of 0.05 to 100 millimolar KCl were complex but similar to those observed in other plant tissues. In contrast, the kinetic data for Cl⁻ and H₂³²PO₄⁻ over the same concentration range were different from those observed for K⁺, and could be accounted for by a single isotherm in the range of 0.05 to 4 millimolar and by an almost linear increase in influx rate above 4 millimolar. The kinetic data for Cl⁻ transport into intact cultured cells were identical in character to those observed for isolated protoplasts. The results support the view that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast.     

Absorption of magnesium and chloride by excised corn root

Absorption characteristics of Mg²⁺ and Cl⁻ were investigated with 5-day-old excised corn (Zea mays) roots. Uptake from both 0.5 and 10 milliequivalents per liter MgCl₂ solutions occurred at steady state rates for the first 6 hours. Inhibition by dinitrophenol and low temperatures established that absorption during this period was metabolically mediated in the absence and presence of Ca²⁺. Absorption isotherms indicated dual mechanisms of Mg²⁺ and Cl⁻ absorption from solutions above 1 milliequivalent per liter. The effect of H⁺ on absorption of Mg²⁺ and Cl⁻ was typical of that generally reported for other plant roots and other ions. In the physiological pH range, Ca²⁺ greatly suppressed the rate of Mg²⁺ absorption but had little effect on Cl⁻. The influence of Ca²⁺ on Mg²⁺ appeared to be noncompetitive and independent of its effect on membrane permeability.     

Calcium uptake by excised maize roots and interactions with alkali cations

Ca²⁺ uptake was studied in short-term experiments using 5-day-old excised maize roots. This tissue readily absorbs Ca²⁺, and inhibition by dinitrophenol and low temperature shows that the process is metabolically mediated. The uptake of Ca²⁺, like that of other cations, is influenced by the counter ion, the pH and concentration of the ambient solution, and the presence of other cations. The rate of uptake from various salts decreases in the following order: NO₃⁻ > Cl⁻ = Br⁻ > SO₄²⁻. K⁺ and H⁺ greatly interfere with Ca²⁺ absorption, while Li⁺ and Na⁺ have only slight effects.