Urea uptake and urease activity in Corynebacterium glutamicum

When Corynebacterium glutamicum is grown with a sufficient nitrogen supply, urea crosses the cytoplasmic membrane by passive diffusion. A permeability coefficient for urea diffusion of 9 × 10^–7 cm s^–1 was determined. Under conditions of nitrogen starvation, an energy-dependent urea uptake system was synthesized. Carrier-mediated urea transport was catalyzed by a secondary transport system linked with proton motive force. With a K _m for urea of 9 μM, the affinity of this uptake system was much higher than the affinity of urease towards its substrate (K _m approximately 55 mM urea). The maximum uptake velocity depended on the expression level and was relatively low [2–3.5 nmol min^–1 (mg dry wt.)^–1]. Received: 11 August 1997 / Accepted: 2 December 1997     

Characterization of a secondary uptake system for l-glutamate in Corynebacterium glutamicum

Abstract A new transport system for the uptake of l-glutamate was characterized in Corynebacterium glutamicum strain Δ glu, in which the previously described binding protein-dependent glutamate uptake system is not present. Kinetic characterization revealed a highly specific secondary transport system, dependent on sodium ions. Glutamate uptake showed Michaelis-Menten kinetics, with a K _m of 0.6 mM and a V _max of 15 nmol min⁻¹ (mg dw)⁻¹. For the co-transported sodium ions, a relatively low K _m of 3.3 mM was determined.     

Kinetics of NH4+ and K+ uptake by ectomycorrhizal fungi. Effect of NH4+ on K+ uptake

NH₄⁺ and K⁺ uptake experiments have been conducted with 3 ectomycorrhizal fungi, originating from Douglas fir (Pseudotsuga menziesii (Mirb.] Franco) stands. At concentrations up to 250 μM, uptake of both NH₄⁺ and K⁺ follow Michaelis-Menten kinetics. Laccaria bicolor (Maire) P. D. Orton, Lactarius rufus (Scop.) Fr. and Lactarius hepaticus Plowr. ap. Boud. exhibit K_m values for NH₄⁺ uptake of 6, 35, and 55 μM, respectively, and K_m values for K⁺ uptake of 24, 18, and 96 μM, respectively. Addition of 100 μM NH₄⁺ raises the K_m of K⁺ uptake by L. bicolor to 35 μM, while the V_max remains unchanged. It is argued that the increase of K_m is possibly caused by depolarization of the plasma membrane. It is not due to a competitive inhibition of K⁺ by NH₄⁺ since the apparent inhibitor constant is much higher than the K_m, for NH₄⁺ uptake. The possibility that NH₄⁺ and K⁺ are taken up by the same carrier can be excluded. The K_m, values for K⁺ uptake in the two other fungi are not significantly affected by 100 μM NH₄⁺. Except for a direct effect of NH₄⁺ on influx of K⁺ into the cells, there may also be an indirect effect after prolonged incubation of the cells in the presence of 100 μM NH₄⁺.     

Puccinellia tenuiflora maintains a low Na+ level under salinity by limiting unidirectional Na+ influx resulting in a high selectivity for K+ over Na+

Puccinellia tenuiflora is a useful monocotyledonous halophyte that might be used for improving salt tolerance of cereals. This current work has shown that P. tenuiflora has stronger selectivity for K⁺ over Na⁺ allowing it to maintain significantly lower tissue Na⁺ and higher K⁺ concentration than that of wheat under short- or long-term NaCl treatments. To assess the relative contribution of Na⁺ efflux and influx to net Na⁺ accumulation, unidirectional ²²Na⁺ fluxes in roots were carried out. It was firstly found that unidirectional ²²Na⁺ influx into root of P. tenuiflora was significantly lower (by 31–37%) than in wheat under 100 and 150 m m NaCl. P. tenuiflora had lower unidirectional Na⁺ efflux than wheat; the ratio of efflux to influx was similar between the two species. Leaf secretion of P. tenuiflora was also estimated, and found the loss of Na⁺ content from leaves to account for only 0.0006% of the whole plant Na⁺ content over 33 d of NaCl treatments. Therefore, it is proposed that neither unidirectional Na⁺ efflux of roots nor salt secretion by leaves, but restricting unidirectional Na⁺ influx into roots with a strong selectivity for K⁺ over Na⁺ seems likely to contribute to the salt tolerance of P. tenuiflora .     

Effect of gibberellic acid on K+ (Rb+) uptake and transport in sunflower roots

Four-week-old sunflower plants ( Helianthus annuus L. cv. Halcón), grown in different nutrient solutions, were used to study the effects of gibberellic acid (GA₃) on K⁺ (Rb⁺) uptake by roots or transport to the shoot. Gibberellic acid application to the nutrient solution did not affect the exudation process of excised roots. When GA₃ was sprayed on leaves 2 to 6 days before excising the roots, the rate of exudation and the K⁺ flux increased. When the exudation study was done keeping the roots in a nutrient solution in which Rb⁺ replaced K⁺, the GA₃ effects were evident also on Rb⁺ uptake and transport. In intact plants, GA₃ increased the Rb⁺ transported to the shoot but did not affect Rb⁺ accumulation in the root. It is suggested that these GA₃ effects can be explained if it is assumed that GA₃ acts on the transport of ions to the xylem vessels.     

Effect of α-Latrotoxin on Acetylcholine Release and Intracellular Ca2+ Concentration in Synaptosomes: Na+-Dependent and Na+-Independent Components

Abstract: We studied the effect of α-latrotoxin (αLTX) on [¹⁴C]acetylcholine ([¹⁴C]ACh) release, intracellular Ca²⁺ concentration ([Ca²⁺]_i), plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. αLTX (10^?10-10^?8M) caused an elevation of the [Ca²⁺]_i as detected by Fura 2 fluorescence and evoked [¹⁴C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na⁺ in the external medium and another that did not. Displacement of Na⁺ by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca²⁺]_i and [¹⁴C]ACh release by αLTX. The Na⁺-dependent component of the αLTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca²⁺]_i and [¹⁴C]ACh release remained in the absence of Na⁺. Both the Na⁺-dependent and the Na⁺-independent components of the αLTX-evoked [¹⁴C]ACh release partly required the presence of either Mg²⁺ or Ca²⁺. The nonneurotransmitter [¹⁴C]choline was released along with [¹⁴C]ACh, but this release did not depend on the presence of either Na⁺ or Ca²⁺, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na⁺-dependent divalent cation entry and (b) Na⁺-independent divalent cation entry, and (2) nonspecific Na⁺- and divalent cation-independent leakage.     

Carrier-mediated acetate uptake in Corynebacterium glutamicum

Acetate is effectively taken up by whole cells of Corynebacterium glutamicum via a specific carrier with a pH optimum of 8. The K _m of acetate uptake was 50 μM and the V _max 25–35 nmol/mg dw min. The activation energy was determined to be 70 kJ/mol. Acetate uptake was competitively inhibited by propionate with a K _i of about 30 μM and blocked by addition of sulfhydryl reagents. The transport activity was clearly dependent on the membrane potential, but independent of the presence of Na⁺-ions. It is concluded that uptake of acetate proceeds by a secondary, proton coupled mechanism.     

The Na+ and K+ Content of Isolated Torpedo Synaptosomes and Its Effect on Choline Uptake

Abstract: The Na⁺ and K⁺ concentrations in isolated Torpedo marmorata synaptosomes were determined. Synaptosomes made according to the method of Israël et al. have high internal Na⁺ (290 MM) and low internal K⁺ (30 mM) concentrations. Modification of the homogenisation media permitted the isolation of synaptosomes which could maintain transmembrane ion gradients (internal Na⁺, 96 mM; K⁺, 81 mM); 0.1 mM-ouabain abolished these gradients. The trans-membrane Na⁺ gradient started to dissipate after 15 min at 20°C. Inclusion of ATP in the homogenisation medium enabled the synaptosomes to maintain the Na⁺ gradient for about 90 min. The presence of these transmembrane ion gradients stimulated choline uptake sevenfold. It is concluded that (a) by selecting the isolation media, Torpedo synaptosomes can be prepared with transmembrane ion gradients; (b) these gradients are ouabain-sensitive and stimulate choline uptake: (c) the synaptosomes require additional ATP to maintain the ion gradients.     

Inhibition by K+ of Na+-Dependent D-Aspartate Uptake into Brain Membrane Saccules

Na+-dependent uptake of dicarboxylic amino acids in membrane saccules, due to exchange diffusion and independent of ion gradients, was highly sensitive to inhibition by K+. The IC50 was 1-2 mM under a variety of conditions (i.e., whole tissue or synaptic membranes, frozen/thawed or fresh, D-[3H]aspartate (10-1000 nM) or L-[3H]glutamate (100 nM), phosphate or Tris buffer, NaCl or Na acetate, presence or absence of Ca2+ and Mg2+). The degree of inhibition by K+ was also not affected on removal of ion gradients by ionophores, or by extensive washing with H2O and reloading of membrane saccules with glutamate and incubation medium in the presence or absence of K+ (3 mM, i.e., IC70). Rb+, NH4+, and, to a lesser degree Cs+, but not Li+, could substitute for K+. [K+] showed a competitive relationship to [Na+]2. Incubation with K+ before or after uptake suggested that the ion acts in part by allowing net efflux, thus reducing the internal pool of amino acid against which D-[3H]aspartate exchanges, and in part by inhibiting the interaction of Na+ and D-[3H]aspartate with the transporter. The current model of the Na+-dependent high-affinity acidic amino acid transport carrier allows the observations to be explained and reconciled with previous seemingly conflicting reports on stimulation of acidic amino acid uptake by low concentrations of K+. The findings correct the interpretation of recent reports on a K+-induced inhibition of Na+-dependent "binding" of glutamate and aspartate, and partly elucidate the mechanism of action.     

Control of Na+ and K+ transport in Spergularia marina I. Transpiration effects

In this paper we begin our study of factors controlling Na⁺ and K⁺ uptake in the halophyte Spergularia marina (L.) Griseb., with emphasis on plants growing at moderate salinity (0.2x sea water). The involvement of transpiration was considered first because of its potential to account for much or all of the transport of ions, and particularly of Na⁺, to the shoot under these growth conditions. Transpiration was constant with time through most of the light period, quickly dropping to 6% of the day time rate at night. ²²Na⁺ uptake, on the other hand, showed much less day/night variation, and relative transport to the shoot was constant. After establishing that transpiration was linearly related to leaf weight, possible transpiration effects were further considered as correlations between leaf weight and transport to the shoot. Under constant, day-time conditions, with linear effects of time and plant size removed, total transport of ²²Na⁺ to the shoot (per plant) was not correlated to leaf weight. A similar result was found when transport was expressed per gram of root, and when partitioning of total label to the shoot was considered. Finally, the correlation was considered between leaf weight and a Na⁺/K⁺ enrichment factor defined as the Na⁺/K⁺ ratio in the leaves divided by that in the roots. This correlation was also insignificant. The results indicate that analysis of control of Na⁺ and K⁺ uptake and transport in this experimental system need not consider effects of transpiration.     

Control of Na+ and K+ transport in Spergularia marina. III. Relationship between ion uptake and growth at moderate salinity

In this paper, we continue our analysis of Na⁺ and K⁺ uptake by mid-vegetative Spergularia marina (L.) Griseb. plants growing on 0.2x sea water medium, with attention to the relationship of ion uptake and growth. In the first part of the paper, growth analysis techniques are used to compare relative growth rates (RGR) and relative accumulation rates (RAR) for Na⁺ and K⁺. Under constant growth conditions, a high correlation between RGR and RAR indicated that growth and accumulation of both ions were well balanced, resulting in Na⁺ and K⁺ concentrations within the plants which were stable after adjustement to the saline medium. The analysis confirmed the existence of a Na⁺ -related growth stimulation in S. marina and an associated increase in the efficiency of K⁺ utilization for growth. When plants were subjected to more rapid salinization and step changes in the light intensity of the growth chamber, RGR and RAR were again similar, even through the discontinuities in growth conditions, suggesting that growth and ion accumulation were co-regulated rather than simply correlated. The growth analysis data were then transformed to give net uptake rates for Na⁺ and K⁺ and the results were compared to those of isotope studies under similar growth conditions. In roots, the rates estimated by the two techniques differed substantially; net uptake rates reflected primarily growth, while isotope studies indicated a substantial ion exchange rate between mature cells and the growth medium. The rates of transport of either Na⁺ or K⁺ to the shoot were very similar using the two estimation techniques. As the rates measured with isotopes were taken from studies lasting at most a few hours, this suggested a very rapid turnover of the upwardly mobile Na⁺ and K⁺ pools in the roots.     

Isoform Specific Reductions in Na+,K+-ATPase Catalytic (α) Subunits in the Nerve of Rats with Streptozotocin-Induced Diabetes

Abstract: Na⁺,K⁺-ATPase activity in nerve is reduced in rats with streptozotocin-induced diabetes; three different isoforms of the α (catalytic) subunit of the enzyme are present in nerve. Using western blot to determine subunit isoform polypeptide levels in sciatic nerve, we found a substantial reduction in α1-isoform polypeptide (88% at 3 weeks, 94% at 8 weeks) after induction of diabetes by streptozotocin. Reductions in α2 and α3 polypeptide were smaller and not statistically significant. The reduction in amount of all three isoform polypeptides in the nerve of 3-week diabetic animals was corrected by administration of insulin. Accumulation of α1 polypeptide at a nerve ligature indicated that rapid transport of that polypeptide in nerve occurs with normal kinetics. The results implicate a specific marked deficit in α1, much more than α2 or α3, catalytic subunit isoform of Na⁺,K⁺-ATPase in the pathogenesis of diabetic neuropathy.     

Sodium-dependent succinate uptake in purple bacterium Ectothiorhodospira shaposhnikovii

Succinate, malate and fumarate uptake in purple sulfur bacterium Ectothiorhodospira shaposhnikovii, strain 1 K MSU, obligatorily depends on the presence of Na⁺. Other monovalent cations such as K⁺, Li⁺, NH₄⁺ could not replace Na⁺. Experiments with energy-depleted cells have shown that succinate uptake against its concentration gradient can be energized by artificially imposed sodium gradients (ΔpNa).An artificial membrane potential (inside negative) inhibited ΔpNa-driven succinate uptake at pH 7.0 but stimulated it at pH 9.0.The results confirm the suggestion that succinate uptake in E. shaposhnikovii is carried out in symport with Na⁺.     

Induction of the Na+/Pi cotransport system in the plasma membrane of Chara corallina requires external Na+ and low levels of Pi

It was shown in previous studies that the giant freshwater alga Chara corallina does not control its Na⁺‐dependent Pi uptake by monitoring the internal Pi concentration and it was hypothesized that Chara may instead detect changes in Pi supply from the environment. The present work investigated the conditions that control the induction and inactivation of high affinity Na⁺/Pi influx in Chara. Withdrawal of Pi from the external medium resulted in a gradual increase in the rate of uptake measured immediately after Pi was resupplied. The increase continued for at least 7 d of starvation. In the initial stages, 0·5 or 1 µm Pi were more effective at inducing transport activity than no Pi, suggesting that low levels of Pi are actually required for induction. The high Na⁺‐dependent Pi uptake observed in Pi‐starved cells was inactivated by treatment with as little as 1 µm Pi over 6 d. External Na⁺ plays a major role in controlling the capacity for Na⁺/Pi cotransport activity, and in the absence of Na⁺, both induction and inactivation were either delayed or abolished. Na⁺ starvation stimulated Na⁺ uptake even though there were no measurable changes in the concentrations of Na⁺, or of K⁺ or Pi in either the vacuole or cytoplasm. It was concluded that both substrate (Pi) and driver ion (Na⁺) are required at adequate concentrations for the induction of the cotransporter. In the case of Pi, it was suggested that passive leakage of Pi from the cell into the apoplast is sufficient for this purpose but that supplementation by up to 1 µm Pi is more effective at the earlier stage. A mechanism for sensing the external supply of Pi is proposed.     

Impairment of Na+,K+-ATPase activity following enterotoxigenic Campylobacter jejuni infection: changes in Na+, Cl− and 3-O-methyl-D-glucose transport in vitro, in rat ileum

Abstract Unidirectional fluxes of Na⁺, Cl⁻ and 3-O-methyl-D-glucose (3-MG) were measured in vitro across Campylobacter jejuni live culture-infected and control rat ileal short-circuited tissues by the Using Chamber technique. Net secretion of Na⁺ and enhanced secretion of Cl⁻ ions was observed in the infected animals ( P < 0.001, n =6) as compared to the net absorption of Na⁺ and marginal secretion of Cl⁻ ions in the control animals. There was a significant decrease in the mucosal-to-serosal fluxes of 3-MG in C. jejuni -infected rat ileum. The specific Na⁺,K⁺-ATPase activity when measured biochemically in the membrane-rich fraction of enterocytes was found to be significantly lower (58%) in the infected group as compared to the control group ( P < 0.001). Our results therefore suggest that infection with an enterotoxigenic C. jejuni inhibits the Na⁺,K⁺-ATPase activity in rat enterocytes. The impairment of Na⁺,K⁺-ATPase activity thus appears to induce a secondary change in Na⁺,Cl⁻ and 3-MG transport in vitro in rat ileum.     

Short term 22Na+ and 42K+ uptake in intact, mid-vegetative plants

Spergularia marina (L.) Griseb. is. a rapidly growing, annual, coastal halophyte. Because of its small size, it is suitable for isotope studies of ion transport well beyond the seedling stage. The purpose of this report is to establish the similarities and differences between ²²Na⁺ and ⁴²K⁺ uptake in S. marina and in more commonly used mesophytic crop species. Vegetative plants were used 18 days after transfer to solution culture. Plants were grown either on Na⁺-free medium or on 0.2 × sea water. ²²Na⁺ uptake was linear with time for several hours. The rate was relatively insensitive to external concentration between 1 and 180 mol Na⁺ m?³, particularly in Na⁺-free plants. Transport to the shoot accounted for 40 to 70% of the total uptake, dependent on salinity but largely independent of time. ⁴²K⁺ uptake decreased with increasing salinity in Na⁺-free plants and increased in 0.2 × sea water plants. Both uptake and transport to the shoot were non-linear with time, upward concavity suggesting recovery from a manipulative and/or osmotic injury. Steady state root contents were compared with predicted contents based on cortical cell electrical potentials using the Nernst equation. Reasonable agreement was found in all cases except Na⁺ content of 0.2 × sea water plants, in which active efflux was indicated. Uptake studies conducted in the presence of chemical modifiers (dicyclohexylcarbodiimide, dinitrophenol and fusicoccin) showed responses of ⁴²K⁺ uptake as expected from studies on agronomic species, and implied the presence of a similar active uptake here despite the appearance of equilibrium. Active Na⁺ uptake was suggested at low Na⁺ levels. We conclude that S. marina is a promising experimental system combining the rapid nutrient acquisition strategy of agionomically important annuals with a high degree of salt tolerance.     

Heterogeneous Na+ Sensitivity of Na+,K+-ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na⁺ sensitivity of whole brain membrane Na⁺,K⁺-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na⁺, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10⁻⁵ and 10⁻⁷ M ), we were able to discriminate Na⁺ sensitivity of Na⁺, K⁺-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na⁺ sensitivity [ K _a of 3.88 ± 0.25 m M Na⁺ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na⁺ sensitivities, a high ( K _a of 4.98 ± 0.2 m M Na⁺ and n of 1.34 ± 0.10) and a low ( K _a of 28 ± 0.5 m M Na⁺ and an n of 1.92 ± 0.18) Na⁺ sensitivity activated above a thresh old (22 ± 0.3 m M Na⁺); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na⁺ sensitivities (apparent K _a values of 3.5 and 20 m M Na⁺). We show that Na⁺ dependence in the absence of ouabain is the result of at least of five Na⁺ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.     

Effects of 2-iodobenzanilide on potassium uptake, H+ extrusion and K+-stimulated ATPase of corn roots

The carboxanilide systemic fungicide 2-iodobenzanilide (2-IB) after 2 h pretreatment at 0.25 m M inhibited K⁺ and SO₄^2- uptake by excised corn roots ( Zea mays L., cv. Dekalb 342) up to ca 70 and 40%, respectively. Proton extrusion from corn roots was also reduced by ca 50% after 1 h contact, and the microsomal K⁺-stimulated ATPase activity from corn roots and pea stems ( Pisum sativum L., cv. Alaska) inhibited by 50 and 72%, respectively. In contrast, the Mg²⁺-ATPase activities of microsomes and mitochondria at pH 6.0 and 8.7, respectively, were unaffected. After 2 h of preincubation with 0.25 m M 2-IB, O₂ consumption by corn roots and pea stems was inhibited by 12 and 18%, respectively. ATP content of corn roots was not altered by 2-IB treatment. Therefore, energy availability "in vivo" was unaffected and the primary effect on corn roots is suggested to be at the plasmalemma ATPase which forms the proton gradient.
With isolated pea stem mitochondria, 0.25 m M 2-IB inhibited O₂ consumption by ca 60% when NADH or malate plus pyruvate were added as substrates; when succinate was used O₂ consumption was unaffected. The mode of action on isolated mitochondria was different from that shown for carboxin and also formerly attributed to the whole class of carboxanilide fungicides.