Uptake of Phenylalanine into Isolated Barley Vacuoles Is Driven by Both Tonoplast Adenosine Triphosphatase and Pyrophosphatase : Evidence for a Hydrophobic l-Amino Acid Carrier System

The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent K_m-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; V_{max app} was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 × 10⁶ vacuoles). Half-maximal transport rates driven with ATP or PPi were reached at 0.5 millimolar ATP or 0.25 millimolar PPi. ATP-driven transport showed a distinct pH optimum at 7.3 while PPi-driven transport reached maximum rates at pH 7.8. Direct measurement of the H⁺-translocating enzyme activities revealed K_{m app} values of 0.45 millimolar for ATPase (EC 3.6.1.3) and 23 micromolar for pyrophosphatase (PPase) (EC 3.6.1.1). In contrast to the coupled amino acid transport, ATPase and PPase activities had relative broad pH optima between 7 to 8 for ATPase and 8 to 9 for PPase. ATPase as well as ATP-driven transport was markedly inhibited by nitrate while PPase and PPi-coupled transport was not affected. The addition of ionophores inhibited phenylalanine transport suggesting the destruction of the electrochemical proton potential difference Δ μH⁺ while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like l-Trp, l-Leu, and l-Tyr was also stimulated by ATP. They seem to compete for the same carrier system. l-Ala, l-Val, d-Phe, and d-Leu did not influence phenylalanine transport suggesting a stereospecificity of the carrier system for l-amino acids having a relatively high hydrophobicity.     

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.     

Density gradient localization of plasma membrane and tonoplast from storage tissue of growing and dormant red beet : characterization of proton-transport and ATPase in tonoplast vesicles

Membranes from homogenates of growing and of dormant storage roots of red beet (Beta vulgaris L.) were centrifuged on linear sucrose gradients. Vanadate-sensitive ATPase activity, a marker for plasma membrane, peaked at 38% to 40% sucrose (1.165-1.175 grams per cubic centimeter) in the case of growing material but moved to as low as 30% sucrose (1.127 grams per cubic centimeter) during dormancy.

A band of nitrate-sensitive ATPase was found at sucrose concentrations of 25% to 28% or less (around 1.10 grams per cubic centimeter) for both growing and dormant material. This band showed proton transport into membrane vesicles, as measured by the quenching of fluorescence of acridine orange in the presence of ATP and Mg²⁺. The vesicles were collected on a 10/23% sucrose step gradient. The phosphate hydrolyzing activity was Mg dependent, relatively substrate specific for ATP (ATP > GTP > UTP > CTP = 0) and increased up to 4-fold by ionophores. The ATPase activity showed a high but variable pH optimum, was stimulated by Cl⁻, but was unaffected by monovalent cations. It was inhibited about 50% by 10 nanomolar mersalyl, 20 micromolar N,N′-dicyclohexylcarbodiimide, 80 micromolar diethylstilbestrol, or 20 millimolar NO₃⁻; but was insensitive to molybdate, vanadate, oligomycin, and azide. Proton transport into vesicles from the 10/23% sucrose interface was stimulated by Cl⁻, inhibited by NO₃⁻, and showed a high pH optimum and a substrate specificity similar to the ATPase, including some proton transport driven by GTP and UTP.

The low density of the vesicles (1.10 grams per cubic centimeter) plus the properties of H⁺ transport and ATPase activity are similar to the reported properties of intact vacuoles of red beet and other materials. We conclude that the low density, H⁺-pumping ATPase of red beets originated from the tonoplast. Tonoplast H⁺-ATPases with similar properties appear to be widely distributed in higher plants and fungi.

     

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.     

Characterization of in vitro proton pumping by microsomal vesicles isolated from corn coleoptiles

Corn (Zea mays L. cv Golden Cross Bantam) coleoptile microsomal vesicles have been isolated which are capable of ATP-driven H⁺-transport as measured by [¹⁴C]methylamine accumulation and quinacrine fluorescence quenching. Formation of the pH gradient in vitro shows a high specificity for ATP·Mg, is temperature-sensitive, exhibits a pH optimum at 7.5, and is inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Of the divalent cations tested, Mn²⁺ is almost as effective as Mg²⁺, while Ca²⁺ is ineffective. Excess divalent cations, particularly Ca²⁺, reduces the pH gradient. H⁺ transport is strongly promoted by anions, especially chloride, while potassium does not affect pump activity. Studies with ³⁶Cl⁻ indicate that ATP-driven H⁺ transport into the vesicles is associated with chloride uptake. Both carbonyl cyanide-m-chlorophenylhydrazone and the anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene, inhibit methylamine accumulation and ³⁶Cl⁻ uptake. Proton pumping is also blocked by diethyl stilbestrol and N,N′-dicyclohexylcarbodiimide, but is insensitive to oligomycin and vanadate. These properties of the pump are inconsistent with either a mitochondrial or plasma membrane origin.     

Proton Gradient Across the Tonoplast of Riccia fluitans as a Result of the Joint Action of Two Electroenzymes

Using pH-sensitive microelectrodes (in vitro) and acridine orange photometry (in vivo), the actions of the two tonoplast phosphatases, the tp-ATPase and the tp-PPase, were investigated with respect to how effectively they could generate a transtonoplast pH-gradient. Under standard conditions the vacuoles of the aquatic liverwort Riccia fluitans have an in vivo pH of 4.7 to 5.0. In isolated vacuoles a maximal vacuolar pH (pH_v) of 4.74 ± 0.1 is generated in the presence of 0.1 millimolar PP_i, but only 4.93 ± 0.13 in the presence of 2.5 millimolar ATP. Both substrates added together approximate the value for PP_i. Cl⁻-stimulates the H⁺-transport driven by the tp-ATPase, but has no effect on the tp-PPase. The transport activity of the tp-ATPase approximates saturation kinetics (K_½ ≈ 0.5 millimolar), whereas transport by the tp-PPase yields an optimum around 0.1 millimolar PP_i. The transtonoplast pH-gradient is dissipated slowly by weak bases, from which a vacuolar buffer capacity of roughly 300 to 400 millimolar/pH_v unit has been estimated. From the free energy (−11.42 kilojoules per mole) for the hydrolysis of PP_i under the given experimental conditions, we conclude that the PPase-stoichiometry (transported H⁺ per hydrolyzed substrate molecule) must be 1, and that in vivo this enzyme works as a H⁺-pump rather than as a pyrophosphate synthetase.     

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.     

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

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

Evidence for an ATP-Dependent Proton Pump on the Golgi of Corn Coleoptiles

Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on sucrose density gradients, and ATP-dependent proton pumping activity was localized by the techniques of [¹⁴C]methylamine uptake and quinacrine fluorescence quenching. Two peaks of proton pumping activity were detected: a light peak (1.07 grams/cubic centimeter) corresponding to the previously characterized tonoplast-type H⁺-ATPase, and a second peak (1.13 grams/cubic centimeter) which coincided with the Golgi markers, latent UDPase, and glucan synthase I. The second peak was lighter than that of the plasma membrane marker, uridine diphosphoglucose-sterol glucosyltransferase (1.16 grams/cubic centimeter) and was not inhibited by vanadate, an inhibitor of the plasma membrane ATPase. The activity was also better correlated with the Golgi cisternae marker, glucan synthase I, than with latent UDPase, a secretory vesicle marker, but a secretory vesicle location cannot be ruled out. The tonoplast-type and Golgi proton pumps were similar in several respects, including a pH optimum at 7.2, stimulation by chloride, inhibition by diethylstilbestrol and N,N′-dicyclohexylcarbodiimide (DCCD), insensitivity to oligomycin and azide, and nucleotide specificity for Mg²⁺-ATP. However, the Golgi H⁺ pump was much less sensitive to nitrate and iodide, and more sensitive to the anion channel blockers, 4-acetamido-4′-isothiocyano-2,2′-stilbene sulfonic acid (SITS) and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) than the tonoplast-type H⁺-pump. The Golgi pump, but not the tonoplast-type pump, was stimulated by valinomycin in the presence of KCl. It is concluded that the Golgi of corn coleoptiles contains a KCl-stimulated H⁺-ATPase which can acidify the interior of Golgi cisternae and associated vesicles.     

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

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

Auxin regulation of a proton translocating ATPase in pea root plasma membrane vesicles

Pea root microsomal vesicles have been fractionated on a Dextran step gradient to give three fractions, each of which carries out ATP-dependent proton accumulation as measured by fluorescence quenching of quinacrine. The fraction at the 4/6% Dextran interface is enriched in plasma membrane, as determined by UDPG sterol glucosyltransferase and vanadate-inhibited ATPase. The vanadate-sensitive phosphohydrolase is not specific for ATP, has a K_m of about 0.23 millimolar for MgATP, is only slightly affected by K⁺ or Cl⁻ and is insensitive to auxin. Proton transport, on the other hand, is more specific for ATP, enhanced by anions (NO₃⁻ > Cl⁻) and has a K_m of about 0.7 millimolar. Auxins decrease the K_m to about 0.35 millimolar, with no significant effect on the V_max, while antiauxins or weak acids have no such effect. It appears that auxin has the ability to alter the efficiency of the ATP-driven proton transport.     

Plant proton pumping pyrophosphatase: the potential for its pyrophosphate synthesis activity to modulate plant growth

Cellular pyrophosphate (PPi) homeostasis is vital for normal plant growth and development. Plant proton‐pumping pyrophosphatases (H⁺‐PPases) are enzymes with different tissue‐specific functions related to the regulation of PPi homeostasis. Enhanced expression of plant H⁺‐PPases increases biomass and yield in different crop species. Here, we emphasise emerging studies utilising heterologous expression in yeast and plant vacuole electrophysiology approaches, as well as phylogenetic relationships and structural analysis, to showcase that the H⁺‐PPases possess a PPi synthesis function. We postulate this synthase activity contributes to modulating and promoting plant growth both in H⁺‐PPase‐engineered crops and in wild‐type plants. We propose a model where the PPi synthase activity of H⁺‐PPases maintains the PPi pool when cells adopt PPi‐dependent glycolysis during high energy demands and/or low oxygen environments. We conclude by proposing experiments to further investigate the H⁺‐PPase‐mediated PPi synthase role in plant growth.     

Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts

Recent experiments show that exogenous NADH increases the O₂ consumption and uptake of inorganic ions into isolated corn (Zea mays L. Pioneer Hybrid 3320) root protoplasts (Lin 1982, Proc Natl Acad Sci USA 79: 3773-3776). A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 millimolar) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles per milligrams protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 millimolar NADH quadrupled V_max of the mechanism I (saturable) component of K⁺ uptake, while K_m was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K⁺ influx and H⁺ efflux, while NADH-stimulated K⁺ uptake was not or only slightly inhibited. p-Chloromercuribenzene-sulfonic acid, N,N′-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H⁺ and K⁺ fluxes. A combination of 10 millimolar fusicoccin and 1.5 millimolar NADH gave an 11-fold increase of K⁺ influx and a more than 3-fold increase of H⁺ efflux. It is concluded that a plasmalemma ATPase is not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the proton channel is an important element in this transport system. Fusicoccin synergistically stimulates the effect of NADH on K⁺ uptake.     

Alteration of transport activity of proton pumps in coleoptile cells during early development stages of maize seedlings

Comparative analysis of the transport activity of proton pumps (plasmalemma H⁺-ATPase, vacuolar H⁺-ATPase, and vacuolar H⁺-pyrophosphatase) in the membrane preparations obtained from coleoptile cells of etiolated maize seedlings (Zea mays L.) was carried out. The highest level of vacuolar pyrophosphatase activity was observed during the early development of coleoptile cells under growth intensification through the elongation. The role of ATPase pumps of tonoplast and plasmalemma in the transport of hydrogen ions increases during further development. The plasmalemma activity in this process is higher. When the growth stops, the activity of proton pumps becomes significantly lower. Nevertheless, their substrate specificity and sensitivity to proton pump inhibitors do not change, which can be an evidence of physiological significance of pumps in the maintenance of cell homeostasis.     

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.

     

Regulation of pyrophosphate levels by H+-PPase is central for proper resumption of early plant development

The synthesis of DNA, RNA, and de novo proteins is fundamental for early development of the seedling after germination, but such processes release pyrophosphate (PPi) as a byproduct of ATP hydrolysis. The over-accumulation of the inhibitory metabolite PPi in the cytosol hinders these biosynthetic reactions. All living organisms possess ubiquitous enzymes collectively called inorganic pyrophosphatases (PPases), which catalyze the hydrolysis of PPi into two orthophosphate (Pi) molecules. Defects in PPase activity cause severe developmental defects and/or growth arrest in several organisms. In higher plants, a proton-translocating vacuolar PPase (H⁺­PPase) uses the energy of PPi hydrolysis to acidify the vacuole. However, the biological implications of PPi hydrolysis are vague due to the widespread belief that the major role of H⁺­PPase in plants is vacuolar acidification. We have shown that the Arabidopsis fugu5 mutant phenotype, caused by a defect in H⁺­PPase activity, is rescued by complementation with the yeast cytosolic PPase IPP1. In addition, our analyses have revealed that increased cytosolic PPi levels impair postgerminative development in fugu5 by inhibiting gluconeogenesis. This led us to the conclusion that the role of H⁺­PPase as a proton-pump is negligible. Here, we present further evidence of the growth-boosting effects of removing PPi in later stages of plant vegetative development, and briefly discuss the biological role of PPases and their potential applications in different disciplines and in various organisms.     

Kinetics of the Vacuolar H-Pyrophosphatase : The Roles of Magnesium, Pyrophosphate, and their Complexes as Substrates, Activators, and Inhibitors

The responses of the vacuolar membrane (tonoplast) proton-pumping inorganic pyrophosphatase (H⁺-PPase) from oat (Avena sativa L.) roots to changes in Mg²⁺ and pyrophosphate (PPi) concentrations have been characterized. The kinetics were complex, and reaction kinetic models were used to determine which of the various PPi complexes were responsible for the observed responses. The results indicate that the substrate for the oat root vacuolar H⁺-PPase is Mg₂PPi and that this complex is also a non-competitive inhibitor. In addition, the enzyme is activated by free Mg²⁺ and competitively inhibited by free PPi. This conclusion differs from that reached in previous studies, in which it was proposed that MgPPi is the substrate for plant vacuolar H⁺-PPases. However, models incorporating MgPPi as a substrate were unable to describe the kinetics of the oat H⁺-PPase. It is demonstrated that models incorporating Mg₂PPi as the substrate can describe some of the published kinetics of the Kalanchoë daigremontiana vacuolar H⁺-PPase. Calculations of the likely concentrations of Mg₂PPi in plant cytoplasm suggest that the substrate binding site of the oat vacuolar H⁺-PPase would be about 70% saturated in vivo.     

Proton-Translocating Inorganic Pyrophosphatase in Red Beet (Beta vulgaris L.) Tonoplast Vesicles

The substrate and ionic requirements of ATP and inorganic pyrophosphate (PPi) hydrolysis by tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were compared with the requirements of ATP-and PPi-dependent proton translocation by the same material. Both ATP hydrolysis and ATP-dependent proton translocation are most stimulated by Cl⁻ and inhibited by NO₃⁻. NaCl and KCl support similar rates of ATP hydrolysis and ATP-dependent proton translocation while K₂SO₄ supports lesser rates for both. PPi hydrolysis and PPi-dependent proton translocation are most stimulated by K⁺. KCl and K₂SO₄ support similar rates of PPi hydrolysis and PPi-dependent proton translocation but NaCl has only a small stimulatory effect on both. Since PPi does not inhibit ATP hydrolysis and ATP does not interfere with PPi hydrolysis, it is inferred that the two phosphohydrolase and proton translocation activities are mediated by different tonoplast-associated enzymes. The results indicate the presence of an energy-conserving proton-translocating pyrophosphatase in the tonoplast of red beet.     

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.