Polyamine uptake in carrot cell cultures

Putrescine and spermidine uptake into carrot (Daucus carota L.) cells in culture was studied. The time course of uptake showed that the two polyamines were very quickly transported into the cells, reaching a maximum absorption within 1 minute. Increasing external polyamine concentrations up to 100 millimolar showed the existence of a biphasic system with different affinities at low and high polyamine concentrations. The cellular localization of absorbed polyamines was such that a greater amount of putrescine was present in the cytoplasmic soluble fraction, while spermidine was mostly present in cell walls. The absorbed polyamines were released into the medium in the presence of increasing external concentrations of the corresponding polyamine or Ca²⁺. The effects of Ca²⁺ were different for putrescine and spermidine; putrescine uptake was slightly stimulated by 10 micromolar Ca²⁺ and inhibited by higher concentrations, while for spermidine uptake there was an increasing stimulation in the Ca²⁺ concentration range between 10 micromolar and 1 millimolar. La³⁺ nullified the stimulatory effect of 10 micromolar Ca²⁺ on putrescine uptake and that of 1 millimolar Ca²⁺ on spermidine uptake. La³⁺ at 0.5 to 1 millimolar markedly inhibited the uptake of both polyamines, suggesting that it interferes with the sites of polyamine uptake. Putrescine uptake was affected to a lesser extent by metabolic inhibitors than was spermidine uptake. It is proposed that the entry of polyamines into the cells is driven by the transmembrane electrical gradient, with a possible antiport mechanism between external and internal polyamine molecule.     

Polyamine Uptake, Kinetics, and Competition among Polyamines and between Polyamines and Inorganic Cations

Polyamine uptake, the kinetics of this uptake, and the competition among polyamines and between polyamines and inorganic cations were studied in petals of Saintpaulia ionantha Wendl. Uptake experiments using ¹⁴C-labeled polyamines were carried out on single petals, at room temperaure (20°C) and in the light. The results show that putrescine, spermidine, and spermine uptake was dependent on the external pH and occurred up to high external polyamine concentrations with K_m values of 8.6, 1.2, and 2.1 millimolar, respectively, with spermidine being the most absorbed at low concentration (17 micromolar). Putrescine and spermidine did not seem to compete for the same site of absorption. Furthermore, putrescine and spermidine uptake was not inhibited by Ca²⁺, Mg²⁺, and K⁺ at the same concentrations (17 micromolar), whereas 1.7 millimolar Ca²⁺ inhibited and K⁺ enhanced spermidine uptake. The intracellular localization of the absorbed putrescine was determined using two different methods. Very little label was found in the apoplast, while most of it was localized in the 98,500g supernatant. According to our data the vacuole, which represents a substantial part of Saintpaulia parenchyma cells, could be a site of putrescine accumulation. 2,4-Dinitrophenol and diethylstilbestrol did not inhibit uptake; however, at 0°C there was a 35% inhibition of spermidine uptake, compared with the controls kept at 20°C as well as a 68% inhibition with 20 millimolar NaSCN.     

Levels of Polyamines and Kinetic Characterization of Their Uptake in the Soybean Pathogen Phytophthora sojae

Polyamines are ubiquitous biologically active aliphatic cations that are at least transiently available in the soil from decaying organic matter. Our objectives in this study were to characterize polyamine uptake kinetics in Phytophthora sojae zoospores and to quantify endogenous polyamines in hyphae, zoospores, and soybean roots. Zoospores contained 10 times more free putrescine than spermidine, while hyphae contained only 4 times as much free putrescine as spermidine. Zoospores contained no conjugated putrescine, but conjugated spermidine was present. Hyphae contained both conjugated putrescine and spermidine at levels comparable to the hyphal free putrescine and spermidine levels. In soybean roots, cadaverine was the most abundant polyamine, but only putrescine efflux was detected. The selective efflux of putrescine suggests that the regulation of polyamine availability is part of the overall plant strategy to influence microbial growth in the rhizosphere. In zoospores, uptake experiments with [1,4-¹⁴C]putrescine and [1,4-¹⁴C]spermidine confirmed the existence of high-affinity polyamine transport for both polyamines. Putrescine uptake was reduced by high levels of exogenous spermidine, but spermidine uptake was not reduced by exogenous putrescine. These observations suggest that P. sojae zoospores express at least two high-affinity polyamine transporters, one that is spermidine specific and a second that is putrescine specific or putrescine preferential. Disruption of polyamine uptake or metabolism has major effects on a wide range of cellular activities in other organisms and has been proposed as a potential control strategy for Phytophthora. Inhibition of polyamine uptake may be a means of reducing the fitness of the zoospore along with subsequent developmental stages that precede infection.     

Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies

For the first time, the ³¹P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 10⁶ vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H⁺ exchanges at the tonoplast. The ³¹P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.     

Cytoplasmic polyamines block the fast-activating vacuolar cation channel

The fast-activating vacuolar (FV) channel dominates the electrical characteristics of the tonoplast at physiological free Ca²⁺ concentrations. Since polyamines are known to increase in plant cells in response to stress, the regulation of FV channels by polyamines was investigated. Patch-clamp measurements were performed on whole barley ( Hordeum vulgare ) mesophyll vacuoles and on excised tonoplast patches. The trivalent polyamine spermidine and the tetravalent polyamine spermine blocked FV channels with K_d≈ 100 μM and K_d≈ 5 μM, respectively. Increasing cytosolic and vacuolar Ca²⁺ had no effect on putrescine and spermidine binding to FV channels but slightly decreased the affinity for spermine. The inhibition of FV channels by all three polyamines was not voltage-dependent. This points to a different mode of binding compared to inward rectifier K⁺ channels and Ca²⁺-permeable glutamate receptor channels from animal cells, which show rectification due to a voltage-dependent block by polyamines. In plant cells, the common polyamines (putrescine, spermidine and spermine) are likely to mediate a salt stress-induced decrease of ion flux across the vacuolar membrane by blocking FV channels.     

Effect of inorganic cations and metabolic inhibitors on putrescine transport in roots of intact maize seedlings

The specificity and regulation of putrescine transport was investigated in roots of intact maize (Zea mays L.) seedlings. In concentration-dependent transport studies, the kinetics for putrescine uptake could be resolved into a single saturable component that was noncompetitively inhibited by increasing concentrations of Ca²⁺ (50 micromolar to 5 millimolar). Similarly, other polyvalent cations, including Mg²⁺ (1.8 millimolar) and La³⁺ (200 micromolar), almost completely abolished the saturable component for putrescine uptake. This suggests that putrescine does not share a common transport system with other divalent or polyvalent inorganic cations. Further characterization of the putrescine transport system indicated that 0.3 millimolar N-ethyl-maleimide had no effect on putrescine uptake, and 2 millimolar p-chloromercuribenzene sulfonic acid only partially inhibited transport of the diamine (39% inhibition). Metabolic inhibitors, including carbonylcyanide-m-chlorphenylhydrazone (20 micromolar) and KCN (0.5 millimolar), also partially inhibited the saturable component for putrescine uptake (V_max reduced 48-60%). Increasing the time of exposure to carbonylcyanide-m-chlorphenylhydrazone from 30 minutes to 2 hours did not significantly increase the inhibition of putrescine uptake. Electrophysiological evidence indicates that the inhibitory effect on putrescine uptake by these inhibitors is correlated to a depolarization of the membrane potential, suggesting that the driving force for putrescine uptake is the transmembrane electrical potential across the plasmalemma.     

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.     

Effects of polyamines on tyrosine hydroxylase activity in adrenals

The ability of polyamines (putrescine, spermidine, and spermine) to modify tyrosine hydroxylase (TH) activity was examined in crude or purified enzyme preparation and in adrenal tissue slices. Polyamines showed biphasic effects on TH activity in vitro at physiological pH 7.0, with an inhibitory effect at low concentrations (<1 mM) and a stimulatory effect at high concentrations. The degree of both inhibition and stimulation produced by polyamines at low and high concentrations, respectively, were proportional to the number of the amino group in the polyamines (putrescine < spermidine < spermine). The degree of inhibition by polyamines was much greater with purified enzyme than with crude enzyme preparations. Tyrosine hydroxylation in situ in adrenal tissue slices was stimulated by polyamines without inhibition at any concentrations tested. This evidence suggests that TH molecules in vivo could interact with polyamines or polyamine-like substances which inhibit the TH activity at physiological concentrations less than 1 mM.     

Membrane transport of sugars and amino acids in isolated protoplasts

A method has been developed for observing membrane transport in isolated protoplasts. Transport of sugars and amino acids has been studied in protoplasts isolated from the mesophyll of Pisum sativum L. That uptake was not due to passive diffusion through damaged membranes was demonstrated by supplying simultaneously two sugar stereoisomers, the one ³H-labeled and the other ¹⁴C-labeled. The protoplast membranes were sufficiently functional to discriminate strongly between these stereoisomers.

To characterize transport the nonmetabolized glucose analogue 3-O-methyl glucose (MeG) and amino acid analogue α-aminoisobutyric acid (AIB) were employed. When uptake was compared per unit of protein as between leaf strips and protoplasts prepared from the same tissue, it was estimated that the protoplasts had retained approximately 40 to 50% of the uptake ability of the whole cells. Uptake of neither MeG nor AIB by protoplasts was linear with time, but the tendency to flatten was more marked for AIB. Addition of Mg-ATP to buffered medium significantly promoted AIB uptake, an effect not ascribable to either chelation or pH. Transport of both MeG and AIB was markedly pH-dependent, uptake falling with rise in pH.

The stimulatory effect of Mg-ATP and the pH dependence confirm that uptake was not due to a diffusional inward “leak” but involved membrane function.

This work demonstrates the feasibility of using isolated protoplasts for membrane transport studies. The potential advantages of using protoplasts for such studies are pointed out.

     

Are Polyamines Transported in Etiolated Peas?

To investigate the possible transport of polyamines and their precursor amino acids, ¹⁴C-labeled putrescine, spermidine, arginine, or lysine were injected into cotyledons of 4-day etiolated pea (Pisum sativum L. cv Alaska) seedlings. After 4 hours the shoot, root, and cotyledons were homogenized and the extracted, dansylated polyamines separated by thin-layer chromatography. Little radioactivity was transported from the cotyledons when [¹⁴C]putrescine or [¹⁴C]spermidine were injected and of the radioactivity in the axis, none could be recovered as polyamines. Injection of [¹⁴C]arginine or [¹⁴C]lysine, on the other hand, led to a significant transport of radioactivity into the axis, of which a large fraction was present in the form of the diamines, putrescine or cadaverine, respectively. These results indicate that polyamines in the growing regions of etiolated pea seedlings probably arise from transport and conversion of amino acid precursors.     

In-situ determination of intracellular concentrations of K+ in barley (Hordeum vulgare L. cv. Kara) using the K+-binding fluorescent dye benzofuran isophthalate

The tetra[acetoxymethyl] ester of the K⁺-binding fluorescent dye benzofuran isophthalate (PBFI-AM) was used to determine changes in intracellular potassium (K⁺) concentrations and to measure net transport of K⁺ in barley (Hordeum vulgare L. cv. Kara) root and leaf protoplasts. When this dye binds to free K⁺ inside the cytoplasm, the fluorescence intensity ratio 340/380 nm increases in direct relation to the K⁺ concentration. Because of a delay in the uptake of dye into the vacuoles, it is possible to determine K⁺ concentrations in the vacuoles and transport of K⁺ from the cytoplasm into the vacuole. The uptake of PBFI-AM in root and leaf protoplasts of barley differed in the absence or presence of external K⁺ and was faster at pH 5.5 than at pH 7.0. The fluorescence intensity of the dye was stable for at least 20 h when the protoplasts were kept at 4°C. In the presence of nigericin, the fluorescence intensity of both cells and protoplasts was linearly related to the external concentration of K⁺ (up to 100 mM).     

Functional analysis of <Emphasis Type="Italic">OsPUT1</Emphasis>, a rice polyamine uptake transporter

Polyamines are nitrogenous compounds found in all eukaryotic and prokaryotic cells and absolutely essential for cell viability. In plants, they regulate several growth and developmental processes and the levels of polyamines are also correlated with the plant responses to various biotic and abiotic stresses. In plant cells, polyamines are synthesized in plastids and cytosol. This biosynthetic compartmentation indicates that the specific transporters are essential to transport polyamines between the cellular compartments. In the present study, a phylogenetic analysis was used to identify candidate polyamine transporters in rice. A full-length cDNA rice clone AK068055 was heterologously expressed in the Saccharomyces cerevisiae spermidine uptake mutant, agp2∆. Radiological uptake and competitive inhibition studies with putrescine indicated that rice gene encodes a protein that functioned as a spermidine-preferential transporter. In competition experiments with several amino acids at 25-fold higher levels than spermidine, only methionine, asparagine, and glutamine were effective in reducing uptake of spermidine to 60% of control rates. Based on those observations, this rice gene was named polyamine uptake transporter 1 (OsPUT1). Tissue-specific expression of OsPUT1 by semiquantitative RT-PCR showed that the gene was expressed in all tissues except seeds and roots. Transient expression assays in onion epidermal cells and rice protoplasts failed to localize to a cellular compartment. The characterization of the first plant polyamine transporter sets the stage for a systems approach that can be used to build a model to fully define how the biosynthesis, degradation, and transport of polyamines in plants mediate developmental and biotic responses.     

Polyamine Metabolism and Osmotic Stress : I. Relation to Protoplast Viability

Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine, and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, andVigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.     

Identification of Functional Amino Acid Residues Involved in Polyamine and Agmatine Transport by Human Organic Cation Transporter 2

Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp⁴²⁷, Glu⁴⁴⁸, Glu⁴⁵⁶, Asp⁴⁷⁵, and Glu⁵¹⁶. In addition, Glu⁵²⁴ and Glu⁵³⁰ were involved in putrescine and spermidine uptake activity, and Glu⁵²⁸ and Glu⁵⁴⁰ were weakly involved in putrescine uptake activity. Furthermore, Asp⁵⁵¹ was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues.     

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.     

Effect of Exogenous Putrescine, Spermidine, and Spermine on K Uptake and H Extrusion through Plasmamembrane in Maize Root Segments

The action of exogenous polyamines (putrescine, spermidine, and spermine) on `washing' and fusicoccin-stimulated K⁺ uptake and H⁺ extrusion through the plasmamembrane in maize (Zea mays L., hybrid line Plenus S 516) root apical segments was studied. The results showed that polyamines inhibit the washing-stimulated K⁺ influx and H⁺ extrusion without interfering with K⁺ uptake and H⁺ extrusion stimulated by fusicoccin. Spermidine appeared to be the most effective in inhibiting K⁺ uptake and H⁺ extrusion while putrescine showed a smaller inhibiting action with respect to the others. The analysis of kinetic constants indicated that the polyamines behave as competitive inhibitors with respect to K⁺.     

Transport of anions in isolated barley vacuoles : I. Permeability to anions and evidence for a cl-uptake system

The anion contents of young barley leaves and of mesophyll protoplasts from the leaves was compared. Anion loss from the protoplasts during isolation was small. Although only about 60% of the leaf cells were mesophyll cells, phosphate and sulfate contents of the mesophyll cells accounted for almost 90% of the leaf contents. Chloride accumulated in the leaf epidermis. The rapid isolation of vacuoles from mesophyll protoplasts permitted the determination of vacuolar ion concentrations. Sodium and nitrate levels were very low in the cytoplasm, and much higher in the vacuole. When barley plants were grown in the presence of low NaCl levels, chloride concentrations were comparable in cytoplasm and vacuole, and similar observations were made with sulfate. Cytoplasmic phosphate concentrations were close to 30 millimolar and potassium concentrations 100 millimolar. During a 30 minute incubation period at room temperature, anion contents of isolated vacuoles decreased considerably. Efflux of NO₃⁻ was faster than that of Cl⁻. Phosphate and sulfate crossed the tonoplast only slowly. 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid partially inhibited the efflux of nitrate and, to a lesser extent, that of chloride. Decreased efflux was also observed in the presence of MgATP. In remarkable contrast, p-chloromercuribenzene sulfonate and HgCl₂ stimulated the efflux of nitrate and chloride, but not of phosphate. Labeled chloride was taken up by isolated vacuoles. The apparent K_m for chloride uptake at low chloride concentrations was 2.3 millimolar. At elevated chloride concentrations, chloride did not display saturation characteristics but, rather, characteristics of a diffusional process. Uptake was stimulated by ATP.     

Amine Accumulation in Acidic Vacuoles Protects the Halotolerant Alga Dunaliella salina Against Alkaline Stress

Amines at alkaline pH induce in cells of the halotolerant alga Dunaliella a transient stress that is manifested by a drop in ATP and an increase of cytoplasmic pH. As much as 300 millimolar NH₄⁺ are taken up by the cells at pH 9. The uptake is not associated with gross changes in volume and is accompanied by K⁺ efflux. Most of the amine is not metabolized, and can be released by external acidification. Recovery of the cells from the amine-induced stress occurs within 30 to 60 minutes and is accompanied by massive swelling of vacuoles and by release of the fluorescent dye atebrin from these vacuoles, suggesting that amines are compartmentalized into acidic vacuoles. The time course of ammonia uptake into Dunaliella cells is biphasic—a rapid influx, associated with cytoplasmic alkalinization, followed by a temperature-dependent slow uptake phase, which is correlated with recovery of cellular ATP and cytoplasmic pH. The dependence of amine uptake on external pH indicates that it diffuses into the cells in the free amine form. Studies with lysed cell preparations, in which vacuoles become exposed but retain their capacity to accumulate amines, indicate that the permeability of the vacuolar membrane to amines is much higher than that of the plasma membrane. The results can be retionalized by assuming that the initial amine accumulation, which leads to rapid vacuolar alkalinization, activates metabolic reactions that further increase the capacity of the vacuoles to sequester most of the amine from the cytoplasm. The results indicate that acidic vacuoles in Dunaliella serve as a high-capacity buffering system for amines, and as a safeguard against cytoplasmic alkalinization and uncoupling of photosynthesis.     

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.     

A Possible Role for Polyamines in the Repression of Growth of Bradyrhizobium japonicum Bacteroids in Soybean Nodules

Soybean plants (Glycine max L. Merr. cv. Tamahomare) accumulatesufficient putrescine and spermidine in their nodules to inhibitthe growth of bacteroids of Bradyrhizobium japonicum strain138NR. Gas-chromatographic analysis showed that the mature nodulesfrom 35-d-old plants contained approximately 1.5 µmoleseach of putrescine and spermidine per g fresh weight. Water-soluble(free) putrescine and spermidine were present at concentrationsof 0.39 and 0.13 µmoles per g fresh weight, respectively.Cadaverine and spermine were not detected in the nodules. Ina yeast-extract mannitol broth at a pH above 7.0, putrescine,cadaverine, spermidine, and spermine at more than 0.5, 0.2,0.05, and 0.05 mM, respectively, inhibited the growth of thebacteroids. The effect of the polyamines was bactericidal athigher concentrations. More than 95% of bacteroids were notable to form colonies on agar plates that contained 0.5 mM spermidineat pH 7.0. The high sensitivity to polyamines was a unique characteristicof the bacteroidform cells of this strain. The bacteroids losttheir sensitivity to the polyamines within 24 hours after theirisolation from nodules. The cultured cells of this strain multipliedin the presence of 2 mM spermidine or spermine. (Received January 28, 1993; Accepted June 14, 1993)