The hyperpolarizing and depolarizing effects of 2,4-dinitrophenol on Ehrlich cells.

The ability of glucose to reverse the effects of dinitrophenol on amino acid uptake in Ehrlich cells is a function of pH. At pH 6.0, the presence of glucose does not reverse the inhibitory action of the uncoupler. Nearly complete restoration occurs with glucose at pH 7.4. At pH 8, the presence of glucose may cause a modest increase in amino acid uptake in presence of dinitrophenol. At all pH values, glucose restores ATP and cellular K+ to the control levels at the same pH. Although the cytoplasmic pH changes with changes in the external pH, the cell interior is more alkaline than the medium near pH 6.0 and more acid than the medium at pH 7.8 even after 45 min incubation at 37 degrees C. With dinitrophenol and in presence of glucose the difference in pH between the medium and the cell is minimal at both pH 6.0 and 7.8.     

K+/H+ antiporter functions as a regulator of cytoplasmic pH in a marine bacterium,Vibrio alginolyticus

The marine bacterium, Vibrio alginolyticus, regulates the cytoplasmic pH at about 7.8 over the pH range 6.0–9.0. By the addition of diethanolamine (a membrane-permeable amine) at pH 9.0, the internal pH was alkalized and simultaneously the cellular K⁺ was released. Following the K⁺ exit, the internal pH was acidified until 7.8, where the K⁺ exit leveled off. The K⁺ exit was mediated by a K⁺/H⁺ antiporter that is driven by the outwardly directed K⁺ gradient and ceases to function at the internal pH of 7.8 and below. The Na⁺-loaded cells assayed in the absence of KCl generated inside acidic ΔpH at alkaline pH due to the function of an Na⁺/H⁺ antiporter, but the internal pH was not maintained at a constant value. At acidic pH range, the addition of KCl to the external medium was necessary for the alkalization of cell interior. These results suggested that in cooperation with the K⁺ uptake system and H⁺ pumps, the K⁺/H⁺ antiporter functions as a regulator of cytoplasmic pH to maintain a constant value of 7.8 over the pH range 6.0–9.0.     

Growth regulation and amino acid transport in epithelial cells: Influence of culture conditions and transformation on A,ASC, and l transport activities

Amino acid transport in Madin-Darby canine kidney (MDCK) cells, grown in a defined medium, was investigated as a function of cell density, exposure to specific growth factors, and transformation. MDCK cells were found to transport neutral amino acids by systems similar to the A, ASC, L, and N systems which have been characterized using other cell lines. Experimental conditions were developed for MDCK cells which allowed independent measurement of A, ASC, and L transport activities. The activity of the L system was measured as Na⁺-independent leucine or methionine uptake at pH 7.4. The activity of the A system was measured as Na⁺-dependent α(methylamino)isobutyric acid (mAIB) uptake at pH 7.4, the activity of the ASC system was measured as Na⁺-dependent alanine uptake in the presence of 0.1 mM mAIB at pH 6.0, and the activity of system N was observed by measuring Na⁺-dependent glutamine uptake at pH 7.4 in the presence of high concentrations of A and ASC system substrates. The L transport system responded minimally to changes in growth state, but Na⁺-dependent amino add transport responded to regulation by growth factors, cell density, and transformation. The activities of the A and ASC systems both decreased at high cell density, but these activities responded dissimilarly under other conditions. The activity of the A system was stimulated by insulin, was inhibited by PGE₁, and was elevated 3–7 fold in the transformed cell line, MDCK-T₁. The activity of the ASC system was slightly stimulated by insulin and by PGE₁, but was unchanged after chemical transformation. Changes in cellular growth were monitored and were found to correlate best with the activity of the A system. These results suggested that MDCK cell growth may be more closely related to the activity of the A than of the ASC system.     

The effect of intracellular pH on the rate of hexose uptake in Chlorella.

The rate of hexose uptake by Chlorella is reduced by uncouplers such as carbonyl cyanide p-trifluoromethoxyphenyl hydrazone or dinitrophenol even before concentration equilibrium is reached. The addition of uncouplers changes the membrane potential and the intracellular pH. The membrane potential does not influence the initial velocity of net sugar uptake, whereas manipulation of the cell pH by means of dimethyloxazolidinedione or by butyric acid uncovered a dramatic influence of cell pH on the rate of hexose uptake: at pH values of 7.5--6.8 maximal rate of uptake is observed but at more acid pH a strong inhibition takes place with virtually total blockage of uptake at pH 6.1. The decrease of cell pH to 6.1 in the presence of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone could therefore account for the decrease in hexose transport rate. It was shown that the intracellular pH as such determines the rate of uptake and not the pH difference between inside and outside; the transport rate did not correlate with delta pH.     

Characteristics of synaptosomal tyrosine uptake in various brain regions: Effect of other amino acids

Tyrosine uptake by rat synaptosomes was maximal after 5–10 min of incubation and at 30°C; uptake was inhibited by dinitrophenol (10^?4 M) or ATP (10^?3 M) and increased by reducing sodium concentrations or increasing calcium or potassium. The best model for uptake is a two-carrier system, in which one carrier shows high-affinity uptake and the other may be diffusional. Both uptake mechanisms are more concentrated in catecholamine-rich brain areas, and are inhibited $\text{in vitro}$ by other large, neutral amino acids. At physiologic amino acid brain concentrations, each system probably carries about half of the tyrosine into the nerve terminal.     

pH-dependent glycine uptake in the presence and absence of sodium ions from rat small intestine

Intestinal uptake of glycine in rats was stimulated 15-20% in the presence of 120 mM Na at pH 6.0 and below but around neutral pH, the amino acid uptake was augmented to 60% compared to that in the Na-free medium. Glycine uptake was 30% more at pH 5.5 compared to that at pH 7.3 in the absence of Na. Kinetic analysis revealed a decrease in Kt for glycine uptake (9.62 mM) at pH 5.5 compared to that at pH 7.3 (Kt = 16.67 mM) with no change in maximal velocity (1.51 mumole/10 min/g tissue) in Na-free buffer. Addition of -SH group reacting reagents to the incubation medium produced 36-58% inhibition of glycine uptake in the presence of Na. However, in absence of Na, inhibition of the order of 21-35% and 8-23% was observed at pH 5.5 and 7.0, respectively. These findings suggest that glycine uptake in rat intestine is influenced by pH and -SH groups are implicated in the process(es).     

Amino acid transport systems of JapaneseParamecium symbiont F36-ZK

Analyses of amino acid transport systems in JapaneseParamecium symbiont F36-ZK were performed using¹⁴C-amino acids. Kinetic analyses of amino acid uptake and competitive experiments revealed three transport systems; a basic amino acid transport system, which catalyzed transport of L-Arg and L-Lys, a general amino acid transport system, which had broad specificity for 19 amino acids (but not L-Arg), and an alanine transport system. These three systems were considered to be capable of active transport. Amino acid-proton symport was indicated by the following data: decreases in pH of the medium observed during L-Ser and L-Ala uptake, and uptake of L-Arg, L-Ser and L-Ala being inhibited by carbonyl cyanide m-chlorophenylhydrazone, sodium azide and vanadate. The optimal pH for uptake of neutral amino acids and L-Arg was around 5 and 5 to 6.5, respectively. Uptake of L-Asp and L-Glu was very sensitive to pH and little uptake of L-Asp was measured above pH 6.0. Amino acid uptake was not inhibited by nitrate or ammonium, and cultured cells with ammonium also possessed constitutive uptake systems.     

The use of fluorescamine as a permeant probe to localize phosphatidylethanolamine in intact friend erythroleukaemic cells

The marine bacterium, Vibrio alginolyticus, regulates the cytoplasmic pH at about 7.8 over the pH range 6.0-9.0. By the addition of diethanolamine (a membrane-permeable amine) at pH 9.0, the internal pH was alkalized and simultaneously the cellular K+ was released. Following the K+ exit, the internal pH was acidified until 7.8, where the K+ exit leveled off. The K+ exit was mediated by a K+/H+ antiporter that is driven by the outwardly directed K+ gradient and ceases to function at the internal pH of 7.8 and below. The Na+-loaded cells assayed in the absence of KCl generated inside acidic delta pH at alkaline pH due to the function of an Na+/H+ antiporter, but the internal pH was not maintained at a constant value. At acidic pH range, the addition of KCl to the external medium was necessary for the alkalization of cell interior. These results suggested that in cooperation with the K+ uptake system and H+ pumps, the K+/H+ antiporter functions as a regulator of cytoplasmic pH to maintain a constant value of 7.8 over the pH range 6.0-9.0.     

Analysis of two chloride requirements for sodium-dependent amino acid and glucose transport by intestinal brush-border membrane vesicles of fish

Intestinal brush border vesicles of a Mediterranean sea fish (Dicentrarchus labrax) were prepared using the Ca²⁺-sedimentation method. The transport of glucose, glycine and 2-aminoisobutyric acid is energized by an Na⁺ gradient ($\text{out > in}$). In addition, amino acid uptake requires Cl^? in the extravesicular medium (2-aminoisobutyric acid more than glycine). This Na⁺- and Cl^?-dependent uptake is electrogenic, since it can be stimulated by negative charges inside the vesicles. The specific Cl^? requirement of glycine and 2-aminoisobutyric acid transport is markedly influenced by pH, a change from 6.5 to 8.4 reducing the role played by Cl^?. In the presence of Cl^?, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 2-aminoisobutyric acid uptake is reduced and its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ is enhanced. Cl^? affects also a non-saturable Na⁺-dependent component of this amino acid uptake. Amino acid transport is also increased by intravesicular Cl^? (2-aminoisobutyric acid less than glycine). This effect is more concerned with glucose uptake, which can be then multiplied by 2.3. A concentration gradient ($\text{in > out}$) as well as the presence of Na⁺ in the incubation medium seems to enter into this requirement. This intravesicular Cl^? effect is not influenced by pH between 6.5 and 8.4.     

Controlled-ph batch butanol-acetone fermentation by low acid producing Clostridium acetobutylicum B18

Summary C. acetobutylicum B18 produced a large amount of butanol over a wide range of pH (4.5–6.0). At pH 6.0 fermentation and cell growth were most active at pH 6.0, and the highest values of glucose consumption rate (4.37 g/L-h), butanol productivity (1.0 g/L-h), butyric acid recycle rate (0.31 g/L-h), and cell growth rate (0.2 h^-1) were obtained. There existed a critical pH between 6.0 and 6.5 above which cells switched to organic acid producing mode. Clostridial stage appeared essential for solvent production by strain B18 but sporulation was not necessary for solvent formation.     

THE EFFECT OF pH ON THE DIVISION RATE OF THE COCCOLITHOPHORID CRICOSPHAERA ELONGATA1

The division rate of Cricosphaera elongata was measured as a function of pH in a medium buffered with the CO₂-bicarbonate-carbonate system. The optimum pH for cell division of the coccolithophorid was 7.8. A change of the partial pressure of CO₂ in the medium from 0.03 to 5% did not affect the division rate. Between pH 6.4 and 7.8 changes in the bicarbonate concentration from 0.1 to 6.0 mm and carbonate concentration from 0.007 to 0.1 mm did not affect the rate of division. At loiv experimental pH, C. elongata was nonmotile and grew in clumps; at higher pH values, it was motile and solitary. Coccoliths were not found covering C. elongata if calcite was soluble in the medium.     

THE UPTAKE OF [3H]GABA BY SLICES OF RAT CEREBRAL CORTEX

—A rapid accumulation of [³H]GABA occurs in slices of rat cerebral cortex incubated at 25° or 37° in a medium containing [³H]GABA. Tissue medium ratios of almost 100:1 are attained after a 60 min incubation at 25°. At the same temperature no labelled metabolites of GABA were found in the tissue or the medium. The process responsible for [³H]GABA uptake has many of the properties of an active transport mechanism: it is temperature sensitive, requires the presence of sodium ions in the external medium, is inhibited by dinitrophenol and ouabain, and shows saturation kinetics. The estimated K_m value for GABA is 2·2 × 10^?5m , and V_max is 0·115 μmoles/min/g cortex. There is only negligible efflux of the accumulated [³H]GABA when cortical slices are exposed to a GABA-free medium. [³H]GABA uptake was not affected by the presence of large molar excesses of glycine, l -glutamic acid, l -aspartic acid, or β-aminobutyrate, but was inhibited in the presence of l -alanine, l -histidine, β-hydroxy-GABA and β-guanidinopropionate. It is suggested that the GABA uptake system may represent a possible mechanism for the inactivation of GABA or some related substance at inhibitory synapses in the cortex.     

Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino acid transport.

The maximum specific growth rate of Streptococcus lactis and Streptococcus cremoris on synthetic medium containing glutamate but no glutamine decreases rapidly above pH 7. Growth of these organisms is extended to pH values in excess of 8 in the presence of glutamine. These results can be explained by the kinetic properties of glutamate and glutamine transport (B. Poolman, E. J. Smid, and W. N. Konings, J. Bacteriol. 169:2755-2761, 1987). At alkaline pH the rate of growth in the absence of glutamine is limited by the capacity to accumulate glutamate due to the decreased availability of glutamic acid, the transported species of the glutamate-glutamine transport system. Kinetic analysis of leucine and valine transport shows that the maximal rate of uptake of these amino acids by the branched-chain amino acid transport system is 10 times higher in S. lactis cells grown on synthetic medium containing amino acids than in cells grown in complex broth. For cells grown on synthetic medium, the maximal rate of transport exceeds by about 5 times the requirements at maximum specific growth rates for leucine, isoleucine, and valine (on the basis of the amino acid composition of the cell). The maximal rate of phenylalanine uptake by the aromatic amino acid transport system is in small excess of the requirement for this amino acid at maximum specific growth rates. Analysis of the internal amino acid pools of chemostat-grown cells indicates that passive influx of (some) aromatic amino acids may contribute to the net uptake at high dilution rates.     

Allosteric Properties of Chorismate Mutase from Quercus pedunculata

Chorismate mutase from Quercus pedunculata Ehrh. leaves has been purified by ammonium sulfate precipitation, molecular sieving and hydroxyapatite chromatography. Some results obtained during the purification suggest the presence, in oak, of two isofunctional forms of the enzyme, the one sensitive, the other insensitive to the action of aromatic amino acids. The regulable form exhibits a molecular weight of about 45,000. It is inhibited by tyrosine and by phenylalanine and is activated by tryptophan. In addition to its activating properties, this latter compound, endowed with a great affinity for the enzyme, reverses the inhibition due to the two other amino acids. The H⁺ concentration of the medium plays an important role in the sensitivity of the enzyme with regard to its effectors. Inhibition by tyrosine and by phenylalanine is maximal at pH 6.5, at which value the two ligands present an identical effect. At alkaline pH values, the rate of inhibition decreases regularly, tyrosine becoming the most effective inhibitor. Activation by tryptophan is particularly acute in a mildly acid medium; at pH 6.5, this effector increases the enzymatic activity threefold. Its action is weak (20% activation) in the optimum pH zone (pH 7.8) and increases towards the more basic pH values. As a result, tryptophan maintains a constant level of enzymatic activity throughout a large pH zone (between 6.5 and pH 9.0). The physiological significance of these results is discussed.     

Effect of pH on the metabolism and fertility of turkey spermatozoa

Oxygen uptake during a 4-h incubation period at 37 degrees C, and the motility of the spermatozoa before and after incubation, increased significantly with increasing pH from 6.3 to 8.8. No interaction between buffer and pH was noticed. In a second series of experiments on the aerobic metabolism of turkey spermatozoa, the effect of the pHs 6.8, 7.3 and 7.8 was studied. Fructose was formed from glucose without regard to the pH of the medium. The glucose consumption, i.e. the glucose disappearance minus fructose formation, the lactic acid accumulation, and the oxidation of glucose and of other substances, were higher, although not always statistically, at pH 7.8 than at pH 6.8. The percentage of fertile eggs during the 3rd week of collection after insemination with fresh semen diluted in the pH 7.8 medium was significantly lower than that with semen diluted in the pH 6.8 or 7.3 media. After 4 h of storage at 15 degrees C, the decrease in the fertility of spermatozoa in the high pH medium was apparent from the 1st week of collection.     

Aluminum Uptake by Neuroblastoma Cells

Aluminum uptake studies in viable neuroblastoma cells were performed. Aluminum uptake was largely dependent on the pH of the suspension medium. At physiological pH values, cells were apparently unable to incorporate detectable amounts of aluminum in the absence of proper mediators. Aluminum uptake was enhanced as the pH decreased, attaining a plateau at about pH 6.0. In experiments with 2 x 10(6) cells/ml, pH 6.0, and 25 microM aluminum in the medium, aluminum incorporation reached saturation at 5 nmol of aluminum/mg of cellular protein, accounting for 60-70% of aluminum added. At pH 6.0, cells showed a large capacity for accumulating aluminum; about 70% of intracellular aluminum was associated with the postmitochondrial fraction. At neutral pH, application of apotransferrin seemed to facilitate aluminum translocation into cells via membrane receptors. Fatty acids were also capable of mediating aluminum uptake at neutral pH, probably by forming aluminum-fatty acid complexes. Low molecular weight aluminum chelators, e.g., citrate, inhibited aluminum uptake. Treatment of cells with energy metabolism blockers had virtually no influence on aluminum uptake, indicative of passive mechanisms. The results suggest that aluminum uptake occurs via different modes dependent on growth conditions, such as medium pH.     

Uptake of glutamine by the scutellum of germinating barley grain

Scutella separated from germinating grains of barley (Hordeum vulgare L. cv Himalaya) took up [¹⁴C]glutamine at an initial rate of about 10 micromoles·gram⁻¹·hour⁻¹ in the standard assay conditions (pH 5, 30°C, 1 millimolar glutamine). Inhibition by unlabeled glutamine and by dinitrophenol indicated that about 95% of the uptake was due to carrier-mediated active transport. The pH optimum of the uptake was 5, and after correction for a nonmediated component the uptake appeared to conform to Michaelis-Menten kinetics with an apparent K_m of about 2 millimolar and a V_max of about 25 micromoles·gram⁻¹·hour⁻¹.

The uptake of glutamine was inhibited by all of the 18 amino acids tested; the mode of inhibition was studied only with proline and was competitive. Eight of the ten amino acids tested at high concentrations appeared to be able to inhibit the mediated uptake of glutamine virtually completely. However, when the inhibitory effect of asparagine was extrapolated to an infinitely high concentration of asparagine, about 24% of the mediated uptake of glutamine remained uninhibited. These results suggest that glutamine is taken up by two (or more) rather unspecific amino acid uptake systems, the minor one having no affinity for asparagine.

Glutamine and alanine could completely inhibit the mediated uptake of 1 millimolar leucine, but about 12% of the mediated uptake appeared to be uninhibitable by asparagine. Furthermore, the ratio of the mediated uptake of glutamine to that of leucine changed from 0.9 to 1.7 between days 1 and 3 of germination. These results give further support for the presence of two unspecific amino acid uptake systems in barley scutella.

     

Symport of proton and sucrose in plasma membrane vesicles isolated from spinach leaves

The mechanism of sucrose transport was investigated in plasma membrane (PM) vesicles isolated from spinach (Spinacia oleracea L.) leaves. PM vesicles were isolated by aqueous two-phase partitioning and were equilibrated in pH 7.8 buffer containing K⁺. The vesicles rapidly accumulated sucrose in the presence of a transmembrane pH gradient (ΔpH) with external pH set at 5.8. The uptake rate was slow at pH 7.8. The K⁺-selective ionophore, valinomycin, stimulated uptake in the presence of a ΔpH, and the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), greatly inhibited ΔpH-dependent sucrose uptake. Addition of sucrose to the vesicles resulted in immediate alkalization of the medium. Alkalization was stimulated by valinomycin, was abolished by CCCP, and was sucrose-specific. These results demonstrate the presence of a tightly coupled H⁺/sucrose symporter in PM vesicles isolated from spinach leaves.