CCCP activation of the reconstituted NaK-pump

Summary In the NaK-ATPase proteoliposomes (PLs), the NaK-pump activity, Na⁺ uptake, and ATP hydrolysis were apparently enhanced by carbonyl cyanidem-chlorophenylhydrazone (CCCP) and other ionophores without ion gradients. These ionophore effects were not cation specific. Without ionophores, the PL's ATPase activity fell to its steady-state value within 3 sec at 15°C. This decrease in activity disappeared in the presence of CCCP. Since CCCP is believed to enhance proton mobility across the lipid bilayer and dissipate membrane potential (V _m ), we postulated that aV _m build-up partially inhibits the PLs by changing the conformation of the NaK-pump, and that CCCP eliminated this partial inhibition. Since this activation required extracellular K⁺ and high ATP concentration in the PLs, CCCP must affect the conversion between the phosphorylated forms of NaK-ATPase (EP); this step has been suggested by Goldschlegger et al. (1987) to be the voltage-sensitive step (J. Physiol. (London) 387:331–355). Although cytoplasmic K⁺ accelerated the change of ADP-and K⁺-sensitive EP (E^*P) to K⁺-sensitive ADP-insensitive EP (E₂P), CCCP did not compete with cytoplasmic K⁺ when cytoplasmic Na⁺ was saturated. When the PLs were phosphorylated with 20 m ATP and 20 m palmitoyl CoA instead of with high concentration of ATP, CCCP increased the E^*P content and decreased the ADP-sensitive K⁺-insensitive EP (E₁P). The results described above suggest that CCCP affects the E₁P to E^*P change in the E₁PE^*PE₂P conversion and that this reaction step is inhibited byV _m .     

Arabidopsis ammonium transporters,AtAMT1;1 and AtAMT1;2, have different biochemical properties and functional roles

We have compared the biochemical properties of two different Arabidopsis ammonium transporters, AtAMT1;1 and AtAMT1;2, expressed in yeast, with the biophysical properties of ammonium transport in planta. Expression of the AtAMT1;1 gene in Arabidopsis roots increased approximately four-fold in response to nitrogen deprivation. This coincided with a similar increase in high-affinity ammonium uptake by these plants. The biophysical characteristics of this high-affinity system (K_m for ammonium and methylammonium of 8 M and 31 M, respectively) matched those of AtAMT1;1 expressed in yeast (K_m for methylammonium of 32 M and K_i for ammonium of 1–10 M). The same transport system was present, although less active, in nitrate-fed roots. Ammonium-fed plants exhibited the lowest rates of ammonium uptake and appeared to deploy a different transporter (K_m for ammonium of 46 M). Expression of AtAMT1;2 in roots was insensitive to changes in nitrogen nutrition. In contrast to AtAMT1;1, AtAMT1;2 expressed in yeast exhibited biphasic kinetics for methylammonium uptake: in addition to a high-affinity phase with a K_m of 36 M, a low-affinity phase with a K_m for methylammonium of 3.0 mM was measured. Despite the presence of a putative chloroplast transit peptide in AtAMT1;2, the protein was not imported into chloroplasts in vitro. The electrophysiological data for roots, together with the biochemical properties of AtAMT1;1 and Northern blot analysis indicate a pre-eminent role for AtAMT1;1 in ammonium uptake across the plasma membrane of nitrate-fed and nitrogen-deprived root cells.     

Stimulation of human cheek cell Na+/H+ antiporter activity by saliva and salivary electrolytes: amplification by nigericin

Proton-dependent, ethylisopropylamiloride (EIPA)-sensitive Na⁺ uptake (Na⁺/H⁺ antiporter) studies were performed to examine if saliva, and ionophores which alter cellular electrolyte balance, could influence the activity of the cheek cell Na⁺/H⁺ antiporter. Using the standard conditions of 1 mmol/1 Na⁺, and a 65:1 (inside:outside) proton gradient in the assay, the uniport ionophores valinomycin (K⁺) and gramicidin (Na⁺) increased EIPA-sensitive Na⁺ uptake by 177% (p < 0.01) and 227% (p < 0.01), respectively. The dual antiporter ionophore nigericin (K⁺-H⁺) increased EIPA-sensitive Na⁺ uptake by 654% (p < 0.01), with maximal Na⁺ uptake achieved by 1 min and at an ionophore concentration of 50 mol/l, with an EC ₅₀ value 6.4 mol/l. Preincubation of cheek cells with saliva or the low molecular weight (MW) components of saliva (saliva activating factors, SAF) for 2 h at 37°C, also significantly stimulated EIPA-sensitive Na⁺ uptake. This stimulation could be mimicked by pre-incubation with 25 mmol/l KCl or K⁺-phosphate buffer. Pre-incubating cheek cells with SAF and the inclusion of 20 mol/1 nigericin in the assay, produced maximum EIPA-sensitive Na⁺ uptake. After pre-incubation with water, 25 mmol/1 K⁺-phosphate or SAF, with nigericin in all assays, the initial rate of proton-gradient dependent, EIPA-sensitive Na⁺ uptake was saturable with respect to external Na⁺ with K_m values of 0.9, 1.7, and 1.8 mmol/l, and V _max values of 13.4, 25.8, and 31.1 nmol/mg protein/30 sec, respectively. With 20 mol/1 nigericin in the assay, Na⁺ uptake was inhibited by either increasing the [K⁺]_o in the assay, with an ID ₅₀ of 3 mmol/l. These results indicate that nigericin can facilitate K⁺ _i exchange for H⁺ _o and the attending re-acidification of the cheek cell amplifies IINa+ uptake via the Na⁺/H⁺ antiporter. The degree of stimulation of proton-dependent, EIPA-sensitive Na⁺ uptake is therefore dependent, in part, on the intracellular K⁺ _i.     

Close coupling between extrusion of H+ and uptake of K+ by barley roots

Extrusion of H⁺ by intact barley (Hordeum vulgare L.) roots was automatically titrated. Simultaneously, uptake of K⁺ into the roots, transport of K⁺ through the roots, and (as a residual term) accumulation of K⁺ within the root tissue were determined. When no monovalent cation was present in the medium the steady rate of H⁺ release was close to zero. Addition of K⁺ stimulated H⁺ extrusion within less than 1 min. The stimulation of H⁺ release was apparently limited only by the movement of K⁺ through the apoplast of the roots. The steady rate of H⁺ extrusion depended on the availability of external K⁺ and saturated at a K⁺ concentration of about 100 mol· dm^-3. Half-maximum rates of net K⁺ uptake and H⁺ extrusion were reached at a K⁺ concentration of about 10 mol·dm^-3. With (slowly absorbable) sulfate as the only anion present, the stoichoimetry between H⁺ release and net K⁺ uptake was one. In conclusion, the uptake of K⁺ across the plasmalemma of the cells of the root cortex is electrically coupled to H⁺ extrusion.     

Multiphasic Morphine Modulation of Substance P Release from Capsaicin-Sensitive Primary Afferent Fibers

Morphine produces a multiphasic modulation of K⁺-evoked substance P release from trigeminal slices and dorsal root ganglion neurons in culture. We now found that the C-fiber stimulant, capsaicin (1 M), evoked release of substance P that was inhibited, enhanced and inhibited by 0.1 nM, 1 M, and 10 M morphine, respectively. This morphine's multiphasic effect was blocked by naloxone (100 nM). Neonatal treatment with capsaicin produced thermal hypoalgesia and abolished the multiphasic effect of morphine on substance P release evoked by 50 mM K⁺. These findings suggest that the multiphasic modulation of substance P release by morphine is dependent on C-type afferents and may be of relevance to nociception.     

Uptake of sewage derived nitrogen by<Emphasis Type="Italic"> Ulva rigida</Emphasis> (Chlorophyceae) in Bahía Nueva (Golfo Nuevo,Patagonia, Argentine)

Uptake kinetics of nitrogen derived from sewage–seawater mixtures (2.5–20% v/v effluent) were determined in the laboratory for Ulva rigida (Chlorophyceae) native from Bahía Nueva (Golfo Nuevo, Patagonia, Argentine). In terms of nitrogen concentration, experimental enrichment levels varied between 53.7 and 362.3M of ammonium and between 0.77 and 6.21M of nitrate+nitrite. Uptake rates were fitted to the Michaelis–Menten equation, with the following kinetic parameters: ammonium: V_max = 591.2molg^–1h^–1, K _s=262.3M, nitrate+nitrite: V _max=12.9molg^–1h^–1, K _s=3.5M). Both nutrients were taken up simultaneously, but ammonium incorporation was faster in all cases. The results show a high capability of Ulva rigida to remove sewage-derived nitrogen from culture media. In the field, most of the nitrogen provided by the effluent would be tied up in algal biomass, supporting low nitrogen levels found at a short distance away from the source.     

Cloning and functional characterization of the high-affinity K<Superscript>+</Superscript> transporter HAK1 of pepper

High-affinity K⁺ uptake in plants plays a crucial role in K⁺ nutrition and different systems have been postulated to contribute to the high-affinity K⁺ uptake. The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K⁺ uptake observed in roots. Pepper plants starved of K⁺ for 3 d showed high-affinity K⁺ uptake (K _m of 6 M K⁺) that was very sensitive to NH and their roots expressed a high-affinity K⁺ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters. When expressed in yeast (Saccharomyces cerevisiae), CaHAK1 mediated high-affinity K⁺ and Rb⁺ uptake with K _m values of 3.3 and 1.9 M, respectively. Rb⁺ uptake was competitively inhibited by micromolar concentrations of NH and Cs⁺, and by millimolar concentrations of Na⁺.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Proton translocation in corn coleoptiles: ATPase or redox chain?

The O₂ dependence of net H⁺ efflux of maize coleoptiles has been investigated. Below 100 M O₂, H⁺ efflux in young (1 cm long) coleoptiles is markedly decreased while old (7 cm long) coleoptiles show a decline only at 10 M O₂. Old coleoptiles show the same decrease in net H⁺ efflux as young ones if treated with fusicoccin. The ratio of alteration of CO₂ production to the change in net proton efflux is about 1:1 at 40–80 M O₂ but not at 10 M O₂. An influx can be observed at 10 M O₂ in young as well as in old coleoptiles if the H⁺ concentration is held at values below pH 6.5. Lower O₂ concentrations lead to an increase of net H⁺ efflux, which might be caused by leaching of organic acids resulting from anaerobic processes, but CO₂ production is not significantly changed at these values. It is proposed that more than one system is responsible for proton translocation across the plasmalemma. One of the systems has a high sensitivity to reduced O₂ concentration which is within the same range as the high K_m of the alternative path.Abbreviation FC fusicoccin     

Influence of nitrogen on the behaviour of nickel in wheat

A glasshouse experiment was conducted to study the effect of Ni on the growth and nutrients concentration in wheat (Triticum aestivum Cv. WH 291) in the presence and absence of applied N as urea. Responses to N application were observed up to 120 g N g^–1 soil. No response to Ni was observed in the dry matter yield of wheat tops (leaves + stem) in the absence of applied N while in the presence of applied N, significant yield increases were obtained at 12.5g Ni g^–1 soil. Nickel was not toxic to wheat up to 50g Ni g^–1 soil in the presence of 120g N g^–1 soil. Nitrogen and Ni concentration in wheat tops and roots increased with increasing levels of applied N and Ni, respectively. Applied Ni had an antagonistic effect on N concentration. Similarly, N reduced the Ni concentration in the wheat tissues. Positive growth responses to Ni were associated with 22 and 15g Ni g^–1 in wheat tops, in the presence of applied N at 60 and 120g N g^–1 soil, while Ni toxicity was associated with 63, 92.5 and 112.5g Ni g^–1 in wheat tops, in the absence and presence of applied N at 60 and 120g N g^–1 soil, respectively.     

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK _m andV _max for GABA uptake, with an increase in theV _max/K _m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na⁺, K⁺-ATPase acting as an ion pump.     

The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

Uptake and K+-stimulated release of [14C]glycine from frog retinal synaptosomal fractions

The uptake of [¹⁴C]glycine and the effect of depolarizing potassium concentrations on its release was investigated in the whole frog retina and its synaptosomal fractions. The uptake of [¹⁴C]glycine in retina and synaptosomal fractions was found to be saturable as well as energy and Na⁺-dependent. TheK _m value for glycine uptake was found to be 46 M for P₂ fraction and 100 M for P₁ fraction, with aV _max of 3.5 and 3.8 nmol/mg protein/min respectively. The release of [¹⁴C]glycine from P₁ and P₂ synaptosomal fractions was markedly increased by raising potassium concentration in the medium, in a partially Ca²⁺-dependent manner. Evoked glycine release was 50% reduced when calcium was omitted from the medium. The K⁺-stimulated release of glycine from P₂ fraction was significantly reduced in the presence of TTX. The cellular origin of the P₁ and P₂ synaptosomal fractions releasing glycine is discussed.     

Accumulation of silver by growing and non-growing populations of Citrobacter intermedius B6

Summary Accumulation of silver is reported for growing and non-growing populations of Citrobacter intermedius B6. In non-growing cultures a maximum uptake of 4.35% (w/w) was observed at an initial silver concentration of 2111.2 mol Ag⁺ l^-1. In contrast the maximum uptake of silver by growing bacteria was 2.81% (w/w) at an effective concentration of silver of 217.8 mol·l^-1. Silver accumulation rates in both resting (460 mol Ag⁺ g^-1 per hour) and continuously grown (41 mol Ag⁺ g^-1 per hour) bacteria are higher than previously reported. Cell fractionation and electron microscopy of continuously grown bacteria indicated that accumulation of silver was associated with the cell envelope, in the form of dense deposits of macromolecular proportions. This observation discounts simple surface adsorption as the process of accumulation in growing cultures.     

Alanine and taurine transport by the gill epithelium of a marine bivalve: Effect of sodium on influx

Summary Marine mussels can accumulate amino acids from seawater into the epithelial cells of the gill against chemical gradients in excess of 5×10⁶ to 1. Uptake of both alanine and taurine into gill tissue isolated fromMytilus californianus was found to be dependent upon Na⁺ in the external solution. Uptake of these amino acids was described by Michaelis-Menten kinetics, and a reduction in external [Na⁺] (from 425 to 213mm) increased the apparent Michaelis constants (alanine, from 8 to 17 m; taurine, from 4 to 39 m) without a significant influence on theJ _max's of these processes. Fivemm harmaline, an inhibitor of Na-cotransport processes in many systems, reduced both alanine and taurine uptake by more than 95%; this inhibition appeared to be competitive in nature, with an apparentK _i of 43 m for the interaction with alanine uptake. Increasing the external [Na⁺] from 0 to 510mm produced a sigmoid activation of alanine and taurine uptake withK _Na's of approximately 325mm. The apparent Hill coefficients for this activation were 7.3 and 7.4 for alanine and taurine, respectively. These data are consistent with uptake mechanisms which require comparatively high concentrations of Na⁺ to activate transport, and which couple several Na⁺ ions to the transport of each amino acid. These characteristics, in conjunction with the previously demonstrated low passive permeability of the apical membrane to amino acids, result in systems capable of i) accumulating amino acids from seawater to help meet the nutritional needs of this animal, and ii) maintaining the high intracellular amino-acid concentrations associated with volume regulation in the gill.     

Transport of branched-chain amino acids in Corynebacterium glutamicum

The transport of branched-chain amino acids was characterized in intact cells of Corynebacterium glutamicum ATCC 13032. Uptake and accumulation of these amino acids occur via a common specific carrier with slightly different affiniteis for each substrate (K _m[Ile]=5.4 M, K _m[Leu]=9.0 M, K _m[Val]=9.5 M). The maximal uptake rates for all three substrates were very similar (0.94–1.30 nmol/mg dw · min). The optimum of amino acid uptake was at pH 8.5 and the activation energy was determined to be 80 kJ/mol. The transport activity showed a marked dependence on the presence of Na⁺ ions and on the membrane potential, but was independent of an existing proton gradient. It is concluded, that uptake of branched-chain amino acid transport proceeds via a secondary active Na⁺-coupled symport mechanism.Abbreviations CCCP Carboxyl cyanide m-chlorophenylhydrazone - dw dry weight - MES 2[N-morpholino]ethanesulfonic acid - mon monensin - nig nigericin - TPP tetraphenylphosphonium bromide - Tris tris[hydroxymethyl]aminomethane - val valinomycin     

Pump-leak sodium fluxes in low salt corn roots

Summary The influx and efflux of sodium from 4-hr washed, low salt corn roots (Zea mays L.) has been studied for characterization of passive and active components. Initial Na⁺ content of the roots is very low, 2.25±0.4 mol/g fresh weight. Na⁺ influx in the presence of 0.2mm Ca²⁺ and 0.002 to 20mm K⁺ is passive (a leak) based upon Goldman-type models, being determined by Na⁺ and cell potential (). Na⁺ was not transported by the K⁺ carrier and influx was unaffected by 50 m dicyclohexylcarbodiimide (DCCD). Permeability of the cells to Na⁺ was of the same order asP _k.Efflux of Na⁺ was by an efficient and rapid active transport system (a pump), thus accounting for the failure of these roots to accumulate high levels of Na⁺. In short-term loading and efflux experiments, internal Na⁺ turnover had a half-time of about 5 min. Sodium efflux was unaffected by DCCD. Net H⁺ flux was zero in the presence of DCCD regardless of sodium efflux, indicating absence of Na⁺/H⁺ antiport. Efflux of Na⁺ was equally rapid into medium containing no Na⁺ and only 0.002mm K⁺. K⁺ influx accounted for less than 4% of Na⁺ efflux, prompting the hypothesis that the Na⁺ (or cation?) efflux pump is the second electrogenic system previously defined based upon electrophysiological measurements.     

Wirkung von K+ auf die Fluxe und den Transport von Na+ in Gerstenwurzeln,K+-stimulierter Na+-Efflux in der Wurzelrinde

Summary Barley roots grown on a nutrient solution containing 1 mM Na⁺ but no K⁺ are capable of a considerable Na⁺ transport via the symplasm of the root and the xylem vessels. K⁺ added to the medium surrounding the root cortex severely inhibits this transport after a lag period at a high rate constant (Fig. 3).It is likely that the fluxes of Na⁺ are changed drastically during this transition from low to high K⁺ status. Although originally limited to steady state fluxes, the extended method of efflux analysis for excised roots (Pitman, 1971) has been applied to the non-steady fluxes which occur upon the addition of K⁺ to the roots. It is shown that besides other changes the efflux of ²²Na⁺ through the cortex of barley roots is stimulated instantaneously (Fig. 5) by the addition of K⁺ and presumably by an influx of K⁺ ions. From this a transient, K⁺-stimulated Na⁺ efflux at the plasmalemma of the cortical cells can be estimated. It amounts to 10.9 moles/g fw · h compared to the control efflux of 3.3 moles/g fw · h without K⁺.The stimulated efflux is attributed to a Na⁺ efflux pump at the plasmalemma and is thus related to the K-Na-selectivity of barley plants. The inhibition of the Na⁺ transport by K⁺ is probably a consequence of this increased efflux of Na⁺ from the symplasm through the root cortex.     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

Ammonium-stimulated,sodium-dependent uptake of glutamine in Bacillus pasteurii

The uptake of glutamine was studied in Bacillus pasteurii DSM 33. Only one uptake system was detected in the concentration range studied (between 1 and 100 M glutamine) which exhibited Michaelis-Menten saturation kinetics, with an apparent K _t of 10.7 (±3.5) M glutamine. The uptake was sodium-dependent (apparent K _t=0.2 mM Na⁺); none of several monovalent cations tested was able to replace sodium in the uptake reaction. Ionophores interfering with proton, sodium or potassium gradients across membranes strongly inhibited uptake of glutamine. Low uptake rates correlating with low potassium content and an acidic cytoplasm were measured in cells grown at high ammonium¹ concentrations. Ammonium and other permeant amines as well as potassium stimulated the uptake reaction in these cells, leading to an increase of up to 100-fold in V _max without affecting the affinity of the uptake system. In cells grown at low concentrations of ammonium, an alkaline cytoplasm and both high glutamine uptake activities and potassium content were measured; the uptake reaction was not further stimulated by permeant amines or potassium in such cells. Growth of the strain was inhibited by Tris at high concentrations; this inhibition was relieved by the addition of increasing amounts of ammonium.Abbreviations CCCP carbonylcyanide-m-chlorphenylhydrazone - DCCD dicyclohexylcarbodiimide This work is dedicated to Prof. Dr. H. Kaltwasser on the occasion of his 60th birthday