Isolation of the braZ gene encoding the carrier for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO.

The braZ gene for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO was isolated and characterized. Determination of the nucleotide sequence showed that the braZ gene comprises 1,311 nucleotides specifying a protein of 437 amino acids. Hydropathy analysis suggested that the product is an integral membrane protein with 12 membrane-spanning segments. The amino acid sequence showed extensive homology to those of the braB and brnQ gene products, branched-chain amino acid carriers of P. aeruginosa and Salmonella typhimurium, respectively. By using the T7 RNA polymerase-promoter system, the braZ gene product was identified as a protein of an apparent Mr of 34,000 on a sodium dodecyl sulfate-polyacrylamide gel. Properties of the transport system encoded by braZ were studied by using P. aeruginosa PAO3537, defective in both the high- and low-affinity branched-chain amino acid transport systems (LIV-I and LIV-II, respectively). The transport system encoded by braZ was found to be another effective branched-chain amino acid transport system in P. aeruginosa PAO and was thus designated as LIV-III. This system is specific for isoleucine and valine, giving the same Km value of 12 microM for these amino acids. The system was found, however, to have a very low affinity for leucine, with a Km value of 150 microM, which contrasts with the substrate specificities of LIV-I and LIV-II.     

Transport systems for branched-chain amino acids in Pseudomonas aeruginosa.

The cells of Pseudomonas aeruginosa showed high activity for leucine transport in the absence of Na+, giving a Km value of 0.34 microM. In the presence of Na+, however, two Km values, 0.37 microM (LIV-I system) and 7.6 microM (LIV-II system), were obtained. The former system seemed to serve not only for the entry of leucine, isoleucine, and valine, but also for that of alanine and threonine, although less effectively. However, the LIV-II system served for the entry of branched-chain amino acids only. The LIV-II system alone was operative in membrane vesicles, for the transport of branched-chain amino acids in membrane vesicles required Na+ and gave single Km values for the respective amino acids. When cells were osmotically shocked, the activity of the LIV-I system decreased, whereas the LIV-II system remained unaffected. The shock fluid from P. aeruginosa cells showed leucine-binding activity with a dissociation constant of 0.25 microM. The specificity of the activity was very similar to that of the LIV-I system. These results suggest that a leucine-binding protein(s) in the periplasmic space may be required for the transport process via the LIV-I system of P. aeruginosa.     

Immunoaffinity purification and reconstitution of sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa.

The gene product of braB encoding the Na+(Li+)-coupled carrier protein for L-leucine, L-isoleucine, and L-valine (LIV-II carrier) of Pseudomonas aeruginosa PML strain was identified and overexpressed using a T7 RNA polymerase/promoter plasmid system. The gene product was pulse-labeled with [35S]methionine as a protein of an apparent Mr of 34,000 on a sodium dodecyl sulfate-polyacrylamide gel. Cell membranes overproducing the LIV-II carrier were solubilized with n-dodecyl beta-D-maltopyranoside. The carrier protein was purified from the detergent extract by two purification steps: (i) immunoaffinity column chromatography using purified polyclonal antibody directed against synthetic 13-mer peptide corresponding to the carboxyl terminus region of the carrier and (ii) subsequent DEAE-cellulose column chromatography. The detergent was replaced by n-octyl beta-D-glucopyranoside prior to the first elution and phospholipid was present during purification. Proteoliposomes reconstituted with the purified LIV-II carrier exhibited Na+ or Li+ concentration gradient-driven transport of leucine, isoleucine, and valine. These results show that the LIV-II carrier was purified to be in a functional form.     

Active transport of nonpolar amino acids in Chromatium vinosum

The photosynthetic purple sulfur bacterium, Chromatium vinosum, takes up the amino acids, L-phenylalanine and L-leucine, via two apparently different electrogenic, H+/amino acid symports. Na+ serves as an allosteric modulator for leucine transport, lowering the Km for leucine from 66 to 15 microM. C. vinosum cells also contain a system that transports both isoleucine and valine. The isoleucine/valine system has the attributes of a H+/amino acid symport at pH less than 7.5 but appears to function as a H+/Na+ (Li+)/amino acid symport at pH greater than or equal to 7.5. Na+ gradients produce an allosteric lowering of the Km values for both isoleucine and valine, from 14 to 7 microM and from 34 to 17 microM, respectively. C. vinosum also accumulates D-alanine in an energy-dependent reaction. The transport process appears to involve the electrogenic cotransport of D-alanine and Na+. The Km value for D-alanine was determined to be 9 microM. Unlike the previously characterized C. vinosum L-alanine/Na+ symport, Na+ gradients did not affect the Km for D-alanine transport. L-Alanine and glycine, but not alpha-aminoisobutyric acid, act as competitive inhibitors for D-alanine transport.     

Third system for neutral amino acid transport in a marine pseudomonad.

Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system.     

Sodium-dependent transport of branched-chain amino acids by a monensin-sensitive ruminal peptostreptococcus

A recently isolated ruminal peptostreptococcus which produced large amounts of branched-chain volatile fatty acids grew rapidly with leucine as an energy source in the presence but not the absence of Na. Leucine transport could be driven by an artificial membrane potential (delta psi) only when Na was available, and a chemical gradient of Na+ (delta uNa+) also drove uptake. Because Na+ was taken up with leucine and a Z delta pH could not serve as a driving force (with or without Na), it appeared that leucine was transported in symport with Na+. The leucine carrier could use Li as well as Na and had a single binding site for Na+. The Km for Na was 5.2 mM, and the Km and Vmax for leucine were 77 microM and 328 nmol/mg of protein per min, respectively. Since valine and isoleucine competitively inhibited (Kis of 90 and 49 microM, respectively) leucine transport, it appeared that the peptostreptococcus used a common carrier for branched-chain amino acids. Valine or isoleucine was taken up rapidly, but little ammonia was produced if they were provided individually. The lack of ammonia could be explained by an accumulation of reducing equivalents. The ionophore, monensin, inhibited growth, but leucine was taken up and deaminated at a slow rate. Monensin caused a loss of K, an increase in Na, a slight increase in delta psi, and a decrease in intracellular pH. The inhibition of growth was consistent with a large decrease in ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium-dependent transport of branched-chain amino acids by a monensin-sensitive ruminal peptostreptococcus.

A recently isolated ruminal peptostreptococcus which produced large amounts of branched-chain volatile fatty acids grew rapidly with leucine as an energy source in the presence but not the absence of Na. Leucine transport could be driven by an artificial membrane potential (delta psi) only when Na was available, and a chemical gradient of Na+ (delta uNa+) also drove uptake. Because Na+ was taken up with leucine and a Z delta pH could not serve as a driving force (with or without Na), it appeared that leucine was transported in symport with Na+. The leucine carrier could use Li as well as Na and had a single binding site for Na+. The Km for Na was 5.2 mM, and the Km and Vmax for leucine were 77 microM and 328 nmol/mg of protein per min, respectively. Since valine and isoleucine competitively inhibited (Kis of 90 and 49 microM, respectively) leucine transport, it appeared that the peptostreptococcus used a common carrier for branched-chain amino acids. Valine or isoleucine was taken up rapidly, but little ammonia was produced if they were provided individually. The lack of ammonia could be explained by an accumulation of reducing equivalents. The ionophore, monensin, inhibited growth, but leucine was taken up and deaminated at a slow rate. Monensin caused a loss of K, an increase in Na, a slight increase in delta psi, and a decrease in intracellular pH. The inhibition of growth was consistent with a large decrease in ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+-dependent transport of large neutral amino acids occurs at the abluminal membrane of the blood-brain barrier

Several Na+-dependent carriers of amino acids exist on the abluminal membrane of the blood-brain barrier (BBB). These Na+-dependent carriers are in a position to transfer amino acids from the extracellular fluid of brain to the endothelial cells and thence to the circulation. To date, carriers have been found that may remove nonessential, nitrogen-rich, or acidic (excitatory) amino acids, all of which may be detrimental to brain function. We describe here Na+-dependent transport of large neutral amino acids across the abluminal membrane of the BBB that cannot be ascribed to currently known systems. Fresh brains, from cows killed for food, were used. Microvessels were isolated, and contaminating fragments of basement membranes, astrocyte fragments, and pericytes were removed. Abluminal-enriched membrane fractions from these microvessels were prepared. Transport was Na+ dependent, voltage sensitive, and inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a particular inhibitor of the facilitative large neutral amino acid transporter 1 (LAT1) system. The carrier has a high affinity for leucine (Km 21 +/- 7 microM) and is inhibited by other neutral amino acids, including glutamine, histidine, methionine, phenylalanine, serine, threonine, tryptophan, and tyrosine. Other established neutral amino acids may enter the brain by way of LAT1-type facilitative transport. The presence of a Na+-dependent carrier on the abluminal membrane capable of removing large neutral amino acids, most of which are essential, from brain indicates a more complex situation that has implications for the control of essential amino acid content of brain.     

Neutral amino acid transport in embryonal carcinoma cells

Neutral amino acid transport was characterized in the pluripotent embryonal carcinoma (EC) cell line, OC15. Ten of the thirteen amino acids tested are transported by all three of the major neutral amino acid transport systems--A, L, and ASC--although one system may make a barely measurable contribution in some cases. The characterization of N-methyl-aminoisobutyric acid (meAIB) transport points to this model amino acid as a definitive substrate for System A transport by OC15 cells. Thus, high concentrations of meAIB can be used selectively to block System A transport, and the transport characteristics of meAIB represent system A transport. Kinetic analysis of System A, with a Km = 0.79mM and Vmax = 14.4 nmol/mg protein/5 min, suggests a single-component transport system, which is sensitive to pH changes. While proline transport in most mammalian cells is largely accomplished through System A, it is about equally divided between Systems A and ASC in OC15 cells, and System A does not contribute at all to proline transport by F9 cells, an EC cell line with limited developmental potential. Kinetic analysis of System L transport, represented by Na+-independent leucine transport, reveals a high-affinity, single-component system. This transport system is relatively insensitive to pH changes and has a Km = 0.0031 mM and Vmax = 0.213 nmol/mg protein/min. The putative System L substrate, 2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH), inhibits Systems A and ASC as well as System L in OC15 cells. Therefore, BCH cannot be used as a definitive substrate for System L in OC15 cells. Phenylalanine is primarily transported by Na+-dependent Systems A and ASC (83% Na+-dependent; 73% System ASC) in OC15 cells, while it is transported primarily by the Na+-independent System L in most other cell types, including early cleavage stage mouse embryos and F9 cells. We have also found this unusually strong Na+-dependency of phenylalanine transport in mouse uterine blastocysts (82% Na+-dependent). There is no evidence for System N transport by OC15 cells, since histidine is transported primarily by a Na+-independent, BCH-inhibitable mechanism.     

Sodium-dependent amino acid transport in reconstituted membrane vesicles from Ehrlich ascites cell plasma membranes

Plasma membranes, isolated from Ehrlich ascites tumor cells, were dissolved in 2% cholate, 4 M urea and then reformed into liposomes upon dialysis at 4 degrees with exogenous phospholipids. Reconstituted vesicles regain the ability to transport amino acids. Na+ was shown to accelerate the uptake of alpha-aminoisobutyrate, phenylalanine, and methionine, but not leucine or epsilon-aminohexanoic acid. With the reconstituted vesicles, methionine, but not leucine, inhibited the uptake of alpha-aminoisobutyrate. An apparent Km value for alpha-aminoisobutyrate uptake of 3.0 mM was obtained. This value is close to that observed with the intact cells and the native membrane vesicles. A Na+ gradient (high Na+ outside) increased alpha-aminoisobutyrate uptake, whereas a reversed gradient (high Na+ inside) increased alpha-aminoisobutyrate efflux. The latter flux was increased by valinomycin, suggesting electrogenic transport. A modest extent of coupling between a Na+ gradient and uphill flow of alpha-aminoisobutyrate was observed.     

Genetic separation of high- and low-affinity transport systems for branched-chain amino acids in Escherichia coli K-12.

The Escherichia coli K-12 mutant strain AE4107 (livH::Mu) is defective in the high-affinity binding protein-mediated uptake system for L-leucine, L-valine, and L-isoleucine (LIV-I). We have used this strain to produce mutations in the residual LIV-II membrane-bound branched-chain amino acid uptake system. Mutants selected for their inability to utilize exogenous L-leucine were found to be defective in the LIV-II system and fell into two classes. One class, represented by strain AE410709 (livP9), showed a complete loss of saturable uptake for L-leucine, L-valine, and L-isoleucine up to 50 muM, and a second class, represented by strain AE4017012 (liv-12), showed a residual component of saturable leucine uptake with increased Km. These mutations, livP9 and liv-12, were closely linked and mapped in the 74 to 78 min region of the E. coli genetic map. Strains constructed so that they lacked both LIV-I and LIV-II transport systems excreted leucine. Strains of the genotype livH+ livP were found to have normal high-affinity binding protein-mediated transport (LIV-I and leucine specific), whereas the low-affinity (LIV-II) transport was completely missing. We concluded from these studies that the high-affinity binding protein-mediated transport systems (LIV-I and leucine specific) can operate independently of the membrane-bound LIV-II system.     

Genetic mapping of bra genes affecting branched-chain amino acid transport in Pseudomonas aeruginosa

Pseudomonas aeruginosa PAO mutants defective in the transport systems for branched-chain amino acids were isolated and characterized. Two mutations in strains selected for trifluoroleucine resistance, braA300 and braB307, were mapped in the met-9020-dcu-9108 and the nar-9011-puuC10 region, respectively. The mutation loci in strains selected for azaleucine resistance, braC310 and bra-311 through bra-314, were all located near the fla genes, with an order of region I fla-bra-region II fla. Strains with braA300 showed a marked reduction in the high-affinity branched-chain amino acid transport system (LIV-I) and a considerable decrease in the lower-affinity system (LIV-II). Strains with braB307 were found to be defective in the LIV-II system. Strains selected for azaleucine resistance were all defective only in the LIV-I system and fell into three phenotypically distinct classes. Strains with braC310 produced a binding protein for leucine, isoleucine, valine, alanine, and threonine (LIVAT-BP) altered in binding ability, indicating that the braC gene is the structural one for the LIVAT-BP. Strains with bra-311 or bra-312 showed a complete loss of production of the LIVAT-BP. Strains with bra-313 or bra-314 produced normal levels of functional LIVAT-BP, suggesting that these mutations are located in a gene(s) other than braC.     

Sodium-dependent neutral amino acid transport by human liver plasma membrane vesicles

The activities of several selected Na(+)-dependent amino acid transporters were identified in human liver plasma membrane vesicles by testing for Na(+)-dependent uptake of several naturally occurring neutral amino acids or their analogs. Alanine, 2-(methylamino)isobutyric acid, and 2-aminoisobutyric acid were shown to be almost exclusively transported by the same carrier, system A. Kinetic analysis of 2-(methylamino)isobutyric acid uptake by the human hepatic system A transporter revealed an apparent Km of 0.15 mM and a Vmax of 540 pmol.mg-1 protein.min-1. Human hepatic system A accepts a broad range of neutral amino acids including cysteine, glutamine, and histidine, which have been shown in other species to be transported mainly by disparate carriers. Inhibition analysis of Na(+)-dependent cysteine transport revealed that the portion of uptake not mediated by system A included at least two saturable carriers, system ASC and one other that has yet to be characterized. Most of the glutamine and histidine uptake was Na(+)-dependent, and the component not mediated by system A constituted system N. The largest portion of glycine transport was mediated through system A and the remainder by system ASC with no evidence for system Gly activity. Our examination of Na(+)-dependent amino acid transport documents the presence of several transport systems analogous to those described previously but with some notable differences in their functional activity. Most importantly, the results demonstrate that liver plasma membrane vesicles are a valuable resource for transport analysis of human tissue.     

Transport and deamination of amino acids by a gram-positive, monensin-sensitive ruminal bacterium.

Strain F, a recently isolated ruminal bacterium, grew rapidly with glutamate or glutamine as an energy source in the presence but not the absence of Na. Monensin, a Na+/H+ antiporter, completely inhibited bacterial growth and significantly reduced ammonia production (85%), but 3,3',4',5-tetrachlorosalicylanide (a protonophore) and valinomycin had little effect on growth or ammonia production. Dicyclohexylcarbodiimide, a H(+)-ATPase, inhibitor had no effect. The kinetics of glutamate and glutamine transport were biphasic, showing unusually high rates at high substrate concentrations. On the basis of low substrate concentrations (less than 100 microM), the Km values for glutamate and glutamine were 4 and 11 microM, respectively. Strain F had separate carriers for glutamate and glutamine which could be driven by a chemical gradient of Na. An artificial delta psi was unable to drive transport even when Na was present. The glutamate carrier had a single binding site for Na with a Km of 21 mM; the glutamine carrier appeared to have more than one binding site, and the Km was 2.8 mM. Neither carrier could use Li instead of Na. Histidine and serine were also rapidly transported by Na-dependent systems, but serine alone did not allow growth even when Na was present. Because exponentially growing cells at pH 6.9 had little delta psi (-3 mV) and a slightly reversed Z delta pH (+17 mV), it appeared that the membrane bioenergetics of strain F were solely dependent on Na circulation.     

Na+-independent transport of basic and zwitterionic amino acids in mouse blastocysts by a shared system and by processes which distinguish between these substrates

Preimplantation mouse blastocysts were found to contain at least three mediated components of Na+-independent amino acid transport. The two less conspicuous components seemed to be selective for either cationic or zwitterionic substrates but were not characterized further or examined for multiple transport activities. L-Leucine and L-lysine competed strongly for uptake by the most conspicuous Na+-independent transport process detected in these conceptuses (referred to as component b0,+), and no further heterogeneity of transport activities was found within this component. A series of inhibitors of various strengths had about the same effect on component b0,+ when either leucine or lysine was the substrate, and uptake of each substrate was not affected significantly by changes in the pH between 6.3 and 8.0. Furthermore, the Ki values for mutually competitive inhibition of transport between leucine and lysine and their Km values for transport via component b0,+ were all on the order of about 100 microM. In addition, the Ki values for competitive inhibition of leucine or lysine uptake by valine were approximately 5 mM in both cases, and alanine appeared to be a similarly weak competitive inhibitor of leucine transport. Based on these results, component b0,+ prefers to interact with bulky amino acids that do not branch at the beta-carbon. Moreover, amino acids that branch at the alpha-carbon, such as the leucine analog 3-amino-endo-bicyclo[3.2.1]octane-3-carboxylic acid, were virtually excluded by this component. The substrate reactivity of component b0,+ is more limited than the Na+-dependent transport system B0,+ in blastocysts which accepts both these branched species and less bulky amino acids relatively well as substrates. Thus, mediated amino acid transport in the mouse trophoblast is clearly distinguishable from that in most other mammalian tissues that have been studied. Not only do component b0,+ and system B0,+ and system B0,+ fail to discriminate strongly between basic and zwitterionic substrates, but their relative reactivity with bicyclic amino acids, such as 3-amino-endo-bicyclo[3.2.1]octane-3-carboxylic acid, is the reverse of transport processes in other cell types where these amino acids react strongly with Na+-independent, but not Na+-dependent, systems.     

Chloride-dependence of amino acid transport in rabbit ileum

Chloride-dependence of influx across the brush-border membrane of distal rabbit ileum was examined for beta-alanine, 2-methylaminoisobutyric acid (MeAIB), leucine, lysine, proline and D-glucose. Influx of leucine at 2 mM and of D-glucose at 0.5 mM was chloride-independent indicating that substitution of isethionate for chloride has no unspecific effect on sodium gradient driven transport processes. In contrast influx of beta-alanine and MeAIB was totally dependent on the presence of chloride ions. In the absence of chloride, proline transport was reduced to 20% of its control level. This remaining transport can be accounted for by the function of the carrier of alpha-amino-monocarboxylic acids. Transport of leucine at 0.1 mM was reduced by absence of chloride. This is in accordance with the observation of leucine transport by the beta-alanine carrier. The kinetics of chloride and sodium activation of transport of MeAIB were examined at 1 mM MeAIB. Chloride activation was characterized by a Hill coefficient of 1 and a K1/2 of 23.5 mM, and sodium activation by a Hill coefficient of 2 and a K1/2 of 51 mM. Thus cotransport of chloride with an imino acid would be compatible with the known rheogenic nature of this transport. This study adds the imino acid carrier and the beta-alanine carrier to the group of chloride-dependent, epithelial amino acid transport systems.     

Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.     

Cation and harmaline interactions with Na(+)-independent dibasic amino acid transport system y+ in human erythrocytes and in erythrocytes from a primitive vertebrate the pacific hagfish (Eptatretus stouti).

Transport systems y+, asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na(+)-dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na+ site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na(+)-site inhibitor) with the same affinity as asc (apparent Ki range 1-4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na(+)-independent, dibasic amino acid transport system y+ accepts neutral amino acids when Na+ or another acceptable cation is also present. This latter observation implies that the y+ translocation site binds Na+ and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y(+)-mediated lysine uptake (5 microM, 20 degrees C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na+ functioning as a competitive inhibitor of transport. Apparent Km and Vmax values for lysine transport at 20 degrees C were 15.2 microM and 183 mumol/l cells per h, respectively, in sucrose medium and 59.4 microM and 228 mumol/l cells per h in Na+ medium. Similar results were obtained with y+ in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), indicating that Na(+)-inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 microM, the IC50 value for Na(+)-inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K+ and guanidinium+, also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y+ translocation site, the presence of 150 mM Na+ increased the harmaline inhibition constant (Ki) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y+, asc and ASC represent a family of closely related transporters with a common evolutionary origin.     

Relationship between Na+-dependent cytoplasmic pH homeostasis and Na+-dependent flagellar rotation and amino acid transport in alkalophilic Bacillus

The cytoplasmic pH homeostasis of alkalophilic Bacillus strains required the presence of Na+ in the medium, and Li+ was found to be equivalently substitutable for Na+. Flagellar rotation and amino acid transport of these bacteria also required Na+ but Li+ was not substitutable for Na+. Na+ concentration of about 1 mM was enough for the cytoplasmic pH homeostasis, while more than 10 mM Na+ was required for the full activities of flagellar rotation and amino acid transport. The addition of 150 mM ethanolamine to the cells at pH 9.6 disrupted the pH homeostasis and increased the cytoplasmic pH close to the external pH. Under this condition, however, flagellar rotation and amino acid transport were not so much affected. Thus, it is clear that flagellar rotation and amino acid transport themselves require the presence of Na+ in the medium, independent of the Na+-dependent cytoplasmic pH homeostasis.