Acid-base regulation during ammonium assimilation in Hydrodictyon africanum

Abstract The acid-base balance during ammonium (used to mean NH ₄⁺ and/or NH₃) assimilation in Hydrodictyon africanum has been measured on cells growing with about 1 mol m^?3 ammonium at an external pH of about 6.5. Measurements made included (1) ash alkalinity (corrected for intracellular ammonium) which yields net organic negative charge, (2) the accumulation of organic N in the cells and (3) the change in extracellular H⁺ (from the pH change and the buffer capacity). These measurements showed that some 0.25 excess organic negative charge (half in the cell wall, half inside the plasmalemma) accumulates per organic N synthesized, while some 1.25H⁺ accumulate in the medium per organic N synthesized. Granted a permeability (PNH₃) of some 10^?3 cm s^?1, and a finite [NH₃] in the cytoplasm of these N-assimilating cells it is likely that most of the ammonium entering these growing cells is as NH ₄⁺. This means that most of the H ⁺ appearing in the medium must have originated from inside the cell and have been subjected to active efflux at the plasmalemma: H⁺ accumulates in the medium equivalent to any NH₃ entry by requilibration from exogenous NH ₄⁺. The cell composition (net organic negative charge, organic N content) is very similar in these ammonium-grown cells to that of NO₃⁺grown cells, suggesting that there is no action of a ‘biochemical pH stat’ during longterm assimilation of NO₃⁺in H. africanum. Short-term experiments were carried out at an external pH of 7.2 in which ammonium at various concentrations were supplied to NO₃⁺-grown cells. There was in all cases a rapid influx followed by a slower uptake; at least at the lower concentrations (less than 100 μmol dm^?3) the net influx was all attributable to NH₄⁺influx via a uniporter, probably partly short-circuited by a passive NH₃ efflux due to intrinsic membrane permeability to NH₃. The net ammonium influx was in all cases associated with H⁺ accumulation in the medium. (1.3-1.7 H ⁺ per ammonium taken up); as in the growth experiments, most of the ammonium taken up was assimilated. Determinations of cytoplasmic pH showed either no effect on, or a slight decrease in, pH during ammonium assimilation; the changes that occurred were in the direction expected for actuating a ‘pH-regulating’ change in H⁺ fluxes.     

Transport of Ammonium and Methylammonium Ions by Azotobacter vinelandii

Ammonium and methylammonium are rapidly taken up by cultures of Azotobacter vinelandii respiring in the presence of succinate. The rate of methylamine uptake increased with external pH from 5.5 to 7.5 but increasing the pH further to 8.5 had little effect on activity, indicating that methylammonium cation rather than uncharged methylamine is the permeant species. The kinetics of methylammonium entry followed the Michaelis-Menten relationship, yielding a K_m of 25 μM and a V_max of 3.8 nmol/min per mg of cell protein. At saturating concentrations ammonium was taken up at rates 30-fold higher than those for methylammonium. Ammonium was a competitive inhibitor of methylammonium uptake and gave an inhibition constant of 1 μM. Ammonium derivatives were inhibitors of methylammonium entry in order of effectiveness: hydrazine > methylhydrazine > formamidine > guanidine > dimethylamine > ethylamine; amides and amino acids did not block uptake. Likewise, metal cations inhibited in the order Tl⁺ > Cs⁺ > Rb⁺, whereas Na⁺, K⁺, and Li⁺ produced no significant effect. Methylammonium uptake was blocked in cells exposed to an uncoupler, p-trifluorome-thoxycarbonyl cyanide-phenyl hydrazone or gramicidin D, but not with dicyclo-hexylcarbodiimide or arsenate. Valinomycin stimulated methylammonium entry into cells in a K⁺-free medium but prevented entry in the presence of 10 mM K⁺. Monensin and nigericin had little effect on transport. These results indicate that methylammonium and ammonium ions enter A. vinelandii electrogenically via a specific transporter.     

Altered S5 and S20 ribosomal proteins in revertants of an alanyl-tRNA synthetase mutant of Escherichia coli

Summary An active transport system specific for ammonium and methylammonium is decribed in wild type cells of Aspergillus nidulans. This system has a Km of less than 5x10^-5 M for ammonium as measured by the uptake of ¹⁵NH⁺ ₄ and a Km of 2x10^-5 M and apparent Vmax of 11 nanomoles/min/mg dry weight for methylammonium, by the uptake of ¹⁴C methylammonium. The system concentrates methylammonium at least 120-fold and is probably regulated by the concentration of internal ammonium.Cells of the mutant strain DER-3 possess a reduced rate of ammonium and methylammonium transport under all conditions tested. DER-3 is a double mutant, one mutation being allelic with meaA8 and designated meaA21, the other is unlinked to meaA and designated mod meaA. The heterozygous diploid DER3/+ has wild type transport, indicating that the mutations are recessive. Cells of the mutant strain amrA1 have impaired transport of ammonium and methylammonium, but only under some conditions. amrA1 is recessive. The possible defects of these mutants are discussed.     

The influence of ammonia in nutrient solution on growth and metabolism of cucumber plants

Summary Shoot yield of cucumber plants grown 18 days in nutrient solution with 0.06 mM NH₃ was decreased. Root yield was diminished at 0.09 mM NH₃ The ammonia treatment caused heavy chlorosis increasing with age of leaves. This chlorosis was not due to any nutrient deficiency. Ammonia also influenced the morphology of roots. They were clearly shorter caused by a much smaller size of root cells.The decrease of yield was linked to a reduction of assimilation occurring not only after a long influence of ammonia lasting 14 days, but also within one hour after starting the NH₃ treatment. The decline of assimilation was probably caused by a higher resistance of stomata against CO₂ influx in leaf tissue as can be concluded from the observation that transpiration was decreased in the same way as assimilation.The effect of ammonia in nutrient solution could also be due to the occurrence of higher NH₃ concentrations in leaf tissue, because both, pH of plant press sap as well as NH₄ concentration of plant tissue, were increased.Furthermore, it is shown that the nitrate content of plant tissue was diminished by ammonia whereas ammonium and amide content were raised. Regulation of nitrate uptake of plants by means of ammonium and amide content of tissue is discussed.     

Different transport mechanisms in plant and human AMT/Rh-type ammonium transporters

The conserved family of AMT/Rh proteins facilitates ammonium transport across animal, plant, and microbial membranes. A bacterial homologue, AmtB, forms a channel-like structure and appears to function as an NH₃ gas channel. To evaluate the function of eukaryotic homologues, the human RhCG glycoprotein and the tomato plant ammonium transporter LeAMT1;2 were expressed and compared in Xenopus oocytes and yeast. RhCG mediated the electroneutral transport of methylammonium (MeA), which saturated with K_m = 3.8 mM at pH_o 7.5. Uptake was strongly favored by increasing the pH_o and was inhibited by ammonium. Ammonium induced rapid cytosolic alkalinization in RhCG-expressing oocytes. Additionally, RhCG expression was associated with an alkali-cation conductance, which was not significantly permeable to NH₄⁺ and was apparently uncoupled from the ammonium transport. In contrast, expression of the homologous LeAMT1;2 induced pH_o-independent MeA⁺ uptake and specific NH₄⁺ and MeA⁺ currents that were distinct from endogenous currents. The different mechanisms of transport, including the RhCG-associated alkali-cation conductance, were verified by heterologous expression in appropriate yeast strains. Thus, homologous AMT/Rh-type proteins function in a distinct manner; while LeAMT1;2 carries specifically NH₄⁺, or cotransports NH₃/H⁺, RhCG mediates electroneutral NH₃ transport.     

Ammonium and Methylammonium Transport in Egeria densa Leaves in Conditions of Different H+ Pump Activity

Previous data in Egeria densa leaves demonstrated a strong inhibitory effect of Cs⁺ on passive K⁺ influx and on K⁺-induced, ATP-dependent electrogenic proton extrusion. In this paper we analyzed, using the same material, the effects of Cs⁺ on ammonium (NH₄⁺) and methylammonium (CH₃NH₃⁺) transport in order to elucidate whether a common transport system for K⁺ and NH₄⁺ could be demonstrated. The effects of Cs⁺ on NH₄⁺- and CH₃NH₃⁺-induced titratable H⁺ extrusion (–ΔH⁺) and on transmembrane electrical potential difference (E_m) in E. densa leaves were analyzed in parallel. All experiments were run either in the absence or presence of fusicoccin, corresponding to low or high H⁺-ATPase activity and membrane hyperpolarization and leading, in this material, to respectively active or passive transport of K⁺. The results suggest the presence in E. densa leaves of two distinct pathways for NH₄⁺ uptake: one in common with NH₄⁺ and (with lower affinity) CH₃NH₃⁺, insensitive to Cs⁺, and a second system, operating at higher H⁺-ATPase activity and E_m hyperpolarization, strongly inhibited by Cs⁺ and impermeable to CH₃NH₃⁺. In agreement with this hypothesis, Xenopus laevis oocytes injected with the KAT1 RNA of Arabidopsis thaliana were permeable to K⁺ and NH₄⁺, but not to CH₃NH₃⁺.     

REVERSIBLE KINETIC MODEL FOR THE SHORT-TERM REGULATION OF METHYLAMMONIUM UPTAKE IN TWO PHYTOPLANKTON SPECIES,DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Methylammonium, an ammonium analog, was used to study the short-term kinetics of ammonium uptake in a diatom, Phaeodactylum tricornutum Bohlin, and a green alga, Dunaliella tertiolecta Butcher. Time courses of methylammonium disappearance were measured over a wide range of initial substrate concentrations for the two species. It was shown that feedback inhibition, described mathematically by a reversible enzyme kinetic model, can be used to explain the data. For the two species, there was good agreement between the kinetic parameters obtained from the analysis of the uptake versus substrate curve and those from the fit of the reversible kinetic model to the time-course data. All time courses of CH₃NH₃⁺ disappearance could be described by constants V_m and k_s. Ammonium time-course data show some similarities to its analog, methylammonium. Our study suggests that the apparent change in V_m and k_s with time measured after the addition of saturating ammonium concentrations reflects an uncoupling between transport and assimilation of the substrate rather than a real change in the kinetic parameters of the transport mechanism.     

Human erythrocyte ammonium transport is mediated by functional interaction of ammonium (RhAG) and anion (AE1) transporters

The ammonia/ammonium (NH₃/NH ₄ ⁺ ) influx into red blood cells (RBCs) is mediated by surface glycoprotein RhAG that forms a structural complex with anion exchanger 1 (AE1, band 3). Owing to the activity of this complex, RBCs exposed to the isosmotic ammonium buffer swell and finally lyse. Isoosmotic NH ₄ ⁺ -containing media alters the pH gradient in RBCs (intracellular alkalosis in response to NH₃/NH ₄ ⁺ influx) and triggers the AE1 activity resulting in redundant chloride and water influx and finally in cell swelling. Here we demonstrate that the ammonia/ammonium transport in human RBCs depends on the pH (pH optimum 7.4 ± 0.1), temperature (Q₁₀ 2.6 ± 0.3), HCO ₃ ^? concentration (EC₅₀ 4.7 ± 0.3 mM), and AE1 function. The data confirm functional interactions between AE1 and RhAG. The initial velocity of cell swelling increased almost 50-fold in the isosmotic ammonium buffer containing 25 mM HCO ₃ ^? (37°C) in comparison to the reaction in the same buffer without HCO ₃ ^? . This indicates that the reaction is facilitated mostly by the carrier proteins, not just owing to the simple diffusion of NH₃ across the erythrocyte membrane. We demonstrate that pH_i reaches its maximum value much faster than the volume increase does. These data suggest that there is no direct correlation between pH_i changes and the influx of NH₃/NH ₄ ⁺ . Taken together, our data show that the RhAG and AE1 complex activity enables erythrocytes to be ammonia/ammonium storage sites in order to maintain the physiological blood ammonia/ammonium equilibrium.     

The role of glutamine in regulation of ammonium transport in Azotobacter vinelandii

Under N₂-fixing conditions, Azotobacter vinelandii expresses a specific transport system for methylammonium (ammonium) [E. M. Barnes, Jr. and P. Zimniak (1981) J. Bacteriol. 146, 512–516]. This activity is decreased markedly by culture of cells in the presence of 10 mm ammonium or 2 mm methylammonium; in both cases, the V_max values for methylammonium uptake were 25% of those of N₂-fixing cells. Mixing experiments with assay medium indicate that transport activity is controlled by intracellular rather than extracellular metabolites. Glutamine synthetase activity of cells cultured with ammonium was 33% that of N₂-fixing cultures, but activity was unaffected by incubation with methylammonium. Thus ammonium transport and ammonium fixation are regulated independently. When ammonium was removed from the medium, cells recovered over 90% of the initial transport activity after 1 h; this recovery was not affected by addition of chloramphenicol. The loss of uptake activity in cells incubated with ammonium or methylammonium correlated with over sixfold increases in intracellular levels of glutamine and γ-glutamylmethylamide, respectively. Recovery of transport was accompanied by similar reductions in pools of these compounds. Over one-half of methylammonium transport activity could be blocked by direct addition of 10 mm glutamine or γ-glutamylmethylamide to transport assays; these concentrations were similar to those observed in vivo. The glutamine analog, 6-diazo-5-oxo-l-norleucine, was the most potent inhibitor found (68% inhibition at 10 μm). These results indicate that the regulation of ammonium transport by ammonium and methylammonium is due to inhibition of the transporter by intracellular γ-glutamyl amides rather than by repression of transporter synthesis.     

NH4 + transport system of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1

NH₄ ⁺ transport system of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 (Vibrio ABE-1) was examined by measuring the uptake of [¹⁴C]methylammonium ion (¹⁴CH₃NH^{3 +}) into the intact cells. ¹⁴CH₃NH₃ ⁺ uptake was detected in cells grown in medium containing glutamate as the sole nitrogen source, but not in those grown in medium containing NH₄Cl instead of glutamate. Vibrio ABE-1 did not utilize CH₃NH₃ ⁺ as a carbon or nitrogen source. NH₄Cl and nonradiolabeled CH₃NH₃ ⁺ completely inhibited ¹⁴CH₃NH₃ ⁺ uptake. These results indicate that ¹⁴CH₃NH₃ ⁺ uptake in this bacterium is mediated via an NH₄ ⁺ transport system and not by a specific carrier for CH₃NH₃ ⁺. The respiratory substrate succinate was required to drive ¹⁴CH₃NH₃ ⁺ uptake and the uptake was completely inhibited by KCN, indicating that the uptake was energy dependent. The electrochemical potentials of H⁺ and/or Na⁺ across membranes were suggested to be the driving forces for the transport system because the ionophores carbonylcyanide m-chlorophenylhydrazone and monensin strongly inhibited uptake activities at pH 6.5 and 8.5, respectively. Furthermore, KCl activated ¹⁴CH₃NH₃ ⁺ uptake. The ¹⁴CH₃NH₃ ⁺ uptake activity of Vibrio ABE-1 was markedly high at temperatures between 0° and 15°C, and the apparent K _m value for CH₃NH₃ ⁺ of the uptake did not change significantly over the temperature range from 0° to 25°C. Thus, the NH₄ ⁺ transport system of this bacterium was highly active at low temperatures. Received: August 1, 1998 / Accepted: October 8, 1998     

A mutant of Chlamydomonas reinhardtii altered in the transport of ammonium and methylammonium

Summary A methylammonium-resistant mutant, named hereafter strain 2170 (ma-1), was isolated for the first time from a eukaryotic phototrophic organism. Mutant 2170 from Chlamydomonas reinhardtii carries a single mendelian mutation which results in a decreased rate of uptake of both ammonium and methylammonium without being affected either in uptake of nitrate or nitrite or any of the tested enzyme activities related to ammonium assimilation. Mutant cells could not use methylammonium as nitrogen source nor excrete ammonium into the medium but they had derepressed nitrate and nitrite reductases when growing in the presence of ammonium. Mutant 2170 also exhibited a diminished methylammonium transport rate in comparison with the wild-type cells. We conclude that mutant 2170 is affected in a transport system responsible for the entrance of both ammonium and methylammonium into the cells.Abbreviations CHES 2-(N-Cyclohexylamino)ethanesulphonic acid - MOPS 3(N-morpholine)propanesulphonic acid     

Identification and Characterization of a PutAMT1;1 Gene from Puccinellia tenuiflora

Nitrogen is one of the most important limiting factors for plant growth. However, as ammonium is readily converted into ammonia (NH₃) when soil pH rises above 8.0, this activity depletes the availability of ammonium (NH₄ ⁺) in alkaline soils, consequently preventing the growth of most plant species. The perennial wild grass Puccinellia tenuiflora is one of a few plants able to grow in soils with extremely high salt and alkaline pH (>9.0) levels. Here, we assessed how this species responds to ammonium under such conditions by isolating and analyzing the functions of a putative ammonium transporter (PutAMT1;1). PutAMT1;1 is the first member of the AMT1 (ammonium transporter) family that has been identified in P. tenuiflora. This gene (1) functionally complemented a yeast mutant deficient in ammonium uptake (2), is preferentially expressed in the anther of P. tenuiflora, and (3) is significantly upregulated by ammonium ions in both the shoot and roots. The PutAMT1;1 protein is localized in the plasma membrane and around the nuclear periphery in yeast cells and P. tenuiflora suspension cells. Immunoelectron microscopy analysis also indicated that PutAMT1;1 is localized in the endomembrane. The overexpression of PutAMT1;1 in A. thaliana enhanced plant growth, and increased plant susceptibility to toxic methylammonium (MeA). Here, we confirmed that PutAMT1;1 is an ammonium-inducible ammonium transporter in P. tenuiflora. On the basis of the results of PutAMT1;1 overexpression in A. thaliana, this gene might be useful for improving the root to shoot mobilization of MeA (or NH₄ ⁺).     

Regulation of NH4 + transport pathways in Pisum arvense roots

The effects of metabolic and protein synthesis inhibitors on NH₄ ⁺ uptake by Pisum arvense plants at low (0.05 mM) and high (1 mM) external ammonium concentration were studied. In short-time experiments cycloheximide decreased the ammonium uptake rate at low level of NH₄ ⁺ and increased the absorption of NH₄ ⁺ from uptake medium containing high ammonium concentration. Arsenate and azide supplied into uptake solutions at low ammonium concentration strongly decreased or completely suppressed the NH₄ ⁺ uptake rate, respectively. When the experiments were carried out at high level of ammonium only azide decreased the uptake rate of NH₄ ⁺ and arsenate stimulated this process. Dinitrophenol very strongly repressed the uptake rate of NH₄ ⁺ at both ammonium concentrations. After removing dinitrophenol from both solutions, neither at low nor high external ammonium level the recovery of NH₄ ⁺ uptake rate was achieved within 150 min or 3 h, respectively. The recovery of NH₄ ⁺ uptake rate after removing azide was observed within 90 min and 3 h at low and high ammonium concentrations, respectively. The regulation of NH₄ ⁺ uptake by some inhibitors at low external ammonium level was investigated using plasma membrane vesicles isolated from roots by two-phase partitioning. Orthovanadate completely suppressed the uptake of NH₄ ⁺ by vesicles and quinacrine decreased the NH₄ ⁺ uptake which 55 suggests that ammonium uptake depends on activities of plasma membrane-bound enzymes. On the other hand, it was found that dinitrophenol completely reduced the NH₄ ⁺ uptake by vesicles. The various effects of inhibitors on ammonium uptake dependent on external ammonium concentration suggest the action of different ammonium transport systems in Pisum arvense roots. The ammonium transport into root cells at low NH₄ ⁺ level requires energy and synthesis of protein in the cytoplasm. The research was supported by grant of KBN No. 6PO4C 068 08     

Amine Transport in Riccia fluitans: Cytoplasmic and Vacuolar pH Recorded by a pH-Sensitive Microelectrode

The cytoplasmic and vacuolar pH and changes thereof in the presence of ammonia (NH₄Cl) and methylamine (CH₃NH₃Cl) have been measured in rhizoid cells of Riccia fluitans by means of a pH-sensitive microelectrode.

On addition of 1 micromolar NH₄Cl, the cytoplasmic pH of 7.2 to 7.4 drops by 0.1 to 0.2 pH units, but shifts to pH 7.8 in the presence of 50 micromolar NH₄Cl or 500 micromolar CH₃NH₃Cl. The pH of the vacuole increases drastically from 4.5 to 5.7 with these latter concentrations. Since a NH₄⁺/CH₃NH₃⁺ uniporter has been demonstrated in the plasmalemma of R. fluitans previously (Felle 1983 Biochim Biophys Acta 602:181-195), the concentration-dependent shifts of cytoplasmic pH are interpreted as results of two processes: first, acidification through deprotonation of the actively transported NH₄⁺; and second, alkalinization through protonation of NH₃ which is taken up to a significant extent from high external concentrations. Furthermore, it is concluded that the determination of intracellular pH by means of methylamine distribution is not a reliable method for eucaryotic systems.

     

Methylammonium uptake by Rhodobacter capsulatus

The ammonium uptake system of Rhodobacter capsulatus B100 was examined using the ammonium analog methylammonium. This analog was not transported when cells were grown aerobically on ammonium. When cultured on glutamate as a nitrogen source, or when nitrogen-starved, cells would take up methylammonium. Therefore, in cells grown under nitrogen-limiting conditions, a second system of ammonium uptake (or a modified form of the first) is present which is distinguished by its capacity for transporting the analog in addition to ammonium. The methylammonium uptake system exhibited saturation kinetics with a K _m of 22 M and a V _max of about 3 nmol per min · mg protein. Ammonium completely inhibited analog transport with a K _i in the range of 1 M. Once inside the cell methylammonium was rapidly converted to -N-methylglutamine; however, a small concentration gradient of methylammonium could still be observed. Kinetic parameters reflect the effects of assimilation.The methylammonium uptake system was temperature and pH dependent, and inhibition studies indicated that energy was required for the system to be operative. A glutamine auxotroph (G29) lacking the structural gene for glutanime synthetase did not accumulate the analog, even when nitrogen starved. The Nif^- mutant J61, which is unable to express nitrogenase structural genes, also did not transport methylammonium, regardless of the nitrogen source for growth. However, the mutant exhibited wild-type ammonium uptake and glutamine synthetase activity. These data suggest that transport of ammonium is required for growth on limited nitrogen and is under the control of the Ntr system in R. capsulatus.Abbreviations CCCP carbonyl cyanide-m-chlorophenyl hydrazone - CHES cyclohexylaminoethanesulfonic acid - DMSO dimethyl sulfoxide - GMAD -N-methylglutamine - GS glutamine synthetase - MES 2-(N-morpholino) ethanesulfonic acid - MSX methionine-Dl-sulfoximine - pCMB p-chloromercuribenzoate - Tricine N-tris(hydroxymethyl)methylglycine     

Uptake of Methylamine by Ulva rigida: Transport of Cations and Diffusion of Free Base

Methylamine¹ is taken up rapidly by disks cut from fronds ofUlva rigida (‘Ulva lactuca’) and can be accumulatedat concentrations several hundred times greater than those inthe bathing medium. At pH 8.0 (the pH of sea-water) the relationshipbetween influx and concentration is normally linear up to 0.1–0.3mM, followed by a second, less steep linear phase, the slopeof which decreases or increases with decreasing or increasingexternal pH. When pH is greater than 9.0, however, net uptakesoon ceases. Methylamine influx is greatly reduced at low temperature,by low concentrations of ammonia and, depending on the lengthof storage of the material, by darkness. Influx is also greatlyreduced when disks are pretreated in solutions containing ammoniaand to a much lesser extent when they are pretreated in methylamine,imidazol or nitrate. Methylamine influx lowers intracellularK⁺, increases Cl^– and has no effect on Na⁺. We suggestthat the first linear phase of influx versus concentration reflectsthe operation of an amine cation porter that is rate-limitedby diffusion of CH₃NH₃⁺ through the external unstirred layer,and that the second phase is due to diffusion of CH₃NH₂ intothe tissue.     

Methylammonium uptake by Rhizobium sp. strain 32H1

We present evidence that methylammonium is transported into cowpea Rhizobium sp. strain 32H1 cells by a membrane carrier whose natural substrate is ammonium. After growth in low (0.2%) oxygen, which is necessary for nitrogen fixation by these cells, respiring rhizobial cells took up [14C]methylammonium to high intracellular levels. Cells grown in atmospheric (21%) oxygen did not take up methylammonium. Uptake (transport plus metabolism) was maximal in cells harvested in the early stationary phase of batch culture and had a distinct pH optimum of 6.5 to 7.0. Uptake was inhibited by metabolic poisons that dissipate the proton motive force or inhibit ATP synthesis. Inhibition of uptake by ammonium and the counterflow phenomenon indicated that ammonium and methylammonium share a transport carrier. Of the methylammonium taken up, about 15% was accumulated to intracellular levels 20 times higher than those in the medium; most of the methylammonium was metabolized to gamma-N-methylglutamine.     

Effects of Exogenously Supplied Ammonium on Root Development of Scots Pine (Pinus sylvestris L.) Seedlings

The effect of potentially toxic concentrations of ammonium on root development of Scots pine seedlings raised on Perlite was investigated during growth periods of 3 or 10 weeks after sowing. It was shown that imbalanced ammonium nutrition led to conspicuous changes of root morphology provided the pH value in the medium was allowed to decrease to 3.9 due to the NH⁺₄-dependent proton excretion into the rhizosphere. Ammonium toxicity could not be observed with seedlings treated either with ammonium nitrate or with ammonium chloride at pH 5.3 ? 6.8. While the supply of NH⁺₄ considerably inhibited root development the biomass production of the shoot was increased. Determination of the endogenous level of ammonium in roots and the leaf whorl exclude a simple causal correlation between ammonium toxicity and accumulated ammonium as has been postulated for herbaceous plants.     

Ammonium uptake kinetics in root and leaf cells of Zostera marina L.

Ammonium uptake rates and the mechanism for ammonium transport into the cells have been analysed in Zostera marina L. In the cells of this species, a proton pump is present in the plasmalemma, which maintains the membrane potential. However, this seagrass shows a high-affinity transport mechanism both for nitrate and phosphate which is dependent on sodium and is unique among angiosperms. We have then analysed if the transport of another N form, ammonium, is also dependent of sodium. First, we have studied ammonium transport at the cellular level by measurements of membrane potentials, both in epidermal root cells and mesophyll cells. And second, we have monitored uptake rates in whole leaves and roots by depletion experiments. The results showed that ammonium is taken up by a high-affinity transport system both in root and leaf cells, although two different of kinetics could be discerned in mesophyll cells (with affinity constants of 2.2 ± 1.1 μM NH₄⁺, in the range 0.01-10 μM NH₄⁺, and 23.2 ± 7.1 μM NH₄⁺, at concentrations between 10 and 500 μM NH₄⁺). However, only one kinetic could be observed in epidermal root cells, which showed a K_m = 11.2 ± 1.0 μM NH₄⁺, considering the whole ammonium concentration range assayed (0.01-500 μM NH₄⁺). The higher affinity of leaf cells for ammonium was consistent with the higher uptake rates observed in leaves, with respect to roots, in depletion experiments at 10 μM NH₄⁺ initial concentration. However, when an initial concentration of 100 μM was assayed, the difference between uptake rates was reduced, but still being higher in leaves. Variations in proton or sodium-electrochemical gradient did not affect ammonium uptake, suggesting that the transport of this nutrient is not driven by these ions and that the ammonium transport mechanism could be different to the transport of nitrate and phosphate in this species.     

Methylammonium uptake by Escherichia coli: evidence for a bacterial NH4+ transport system.

Energy-dependent concentrative uptake of ¹⁴CH₃NH₃⁺ by cells of $\text{Escherichia coli}$ provides preliminary evidence for one or more transport systems for NH₄⁺ uptake. NH₄⁺, but not glutamic acid, inhibited the uptake of ¹⁴CH₃NH₃⁺. Varying the pH for the uptake assays exposed two apparent systems: one maximally functioning at pH 7 that was strongly inhibited by cyanide or by the uncoupler $\text{m}$-chlorophenyl carbonylcyanide hydrazone and another maximally functioning at pH 9 and resistant to cyanide or $\text{m}$-chlorophenyl carbonylcyanide hydrazone. Kinetic analysis showed considerable experimental variability from day to day. Often simple Michaelis-Menten kinetics were not followed, but NH₄⁺ was reproducibly a stronger inhibitor of uptake of ¹⁴CH₃NH₃⁺ than was nonradioactive CH₃NH₃⁺.