Elucidation of an A and L system for amino acid transport in the human lymphoblast using a membrane filtration technique.

Optimum conditions have been established for the measurement of amino acid transport by human lymphoblastoid cell lines using a membrane-filtration technique. The parameters we found to be important for the reproducibility of the method are: the types and combination of filters, the strength of the vacuum applied to the filters and the density of the cultures at the time of harvesting and during uptake and filtration. We found that bovine serum albumin added to phosphate buffered saline (PBS) glucose in which the cells are washed, resuspended and assayed is essential for the maintenance of viability, the prevention of clumping and the retention of the accumulated amino acid. Using this procedure we have characterized two transport systems for the neutral amino acids; an A and an L system, which are similar but not identical to the A and L systems characterized in rodent cell lines. These A and L systems have characteristically lower Km's and Vm's for alanine and phenylalanine, when compared to rodent cell lines. In addition, we find alpha-AIB to be a poor competitor of alanine and phenylalanine uptake.     

Role of the membrane potential in serum-stimulated uptake of amino acid in a diploid human fibroblast

The Na⁺-dependent accumulation of α-aminoisobutyric acid (AIB), measured in normal growing and quiescent (serum-deprived) HSWP cells (human diploid fibroblast), was found to be twofold higher (AIB_in/AIB_out = 20–25) under the normal growing conditions. Serum stimulation of quiescent cells increases their AIB concentrating capacity by approximately 70% within 1 hr. These observations suggest that the driving forces for AIB accumulation may be reversibly influenced by the serum concentration of the growth medium. Addition of valinomycin (Val) to cells preequilibrated with AIB causes an enhanced accumulation of AIB, suggesting that the membrane potential can serve as a driving force for AIB accumulation. After preequilibration with AIB in 6 mM K⁺, transfer to 94 mM K⁺ with Val results in a marked and rapid net loss of AIB. The effect of Val on the accumulation of AIB is greatest in quiescent cells, with the intracellular AIB concentrations reaching those seen both in Val-stimulated normal cells and in Val-stimulated serum-stimulated cells. By adjusting [K⁺]₀, in the presence of Val, the membrane potential of growing cells can be matched to that of quiescent cells or vice versa. When this is done, the two accumulate AIB to the same extent. Hence the AIB accumulating capacity is characteristic of the membrane potential rather than of the growth state. In summary, these data suggest that the accumulation of AIB in HSWP cells is influenced by changes in membrane potential and that a serum-associated membrane hyperpolarization could be responsible for the increased capacity for AIB accumulation in serumstimulated cells.     

Multiple transport pathways for neutral amino acids in rabbit jejunal brush border vesicles

Summary Amino acids enter rabbit jejunal brush border membrane vesicles via three major transport systems: (1) simple passive diffusion; (2) Na-independent carriers; and (3) Na-dependent carriers. The passive permeability sequence of amino acids is very similar to that observed in other studies involving natural and artificial membranes. Based on uptake kinetics and cross-inhibition profiles, at least two Na-independent and three Na-dependent carrier-mediated pathways exist. One Na-independent pathway, similar to the classical L system, favors neutral amino acids, while the other pathway favors dibasic amino acids such as lysine. One Na-dependent pathway primarily serves neutrall-amino acids including 2-amino-2-norbornanecarboxylic acid hemihydrate (BCH), but not -alanine or -methylaminoisobutyric acid (MeAIB). Another Na-dependent route favors phenylalanine and methionine, while the third pathway is selective for imino acids and MeAIB. Li is unable to substitute for Na in these systems. Cross-inhibition profiles indicated that none of the Na-dependent systems conform to classical A or ACS paradigms. Other notable features of jejunal brush border vesicles include (1) no -alanine carrier, and (2) no major proline/glycine interactions.     

Amino acid composition analysis of human secondary transport proteins and implications for reliable membrane topology prediction

Secondary transporters in humans are a large group of proteins that transport a wide range of ions, metals, organic and inorganic solutes involved in energy transduction, control of membrane potential and osmotic balance, metabolic processes and in the absorption or efflux of drugs and xenobiotics. They are also emerging as important targets for development of new drugs and as target sites for drug delivery to specific organs or tissues. We have performed amino acid composition (AAC) and phylogenetic analyses and membrane topology predictions for 336 human secondary transport proteins and used the results to confirm protein classification and to look for trends and correlations with structural domains and specific substrates and/or function. Some proteins showed statistically high contents of individual amino acids or of groups of amino acids with similar physicochemical properties. One recurring trend was a correlation between high contents of charged and/or polar residues with misleading results in predictions of membrane topology, which was especially prevalent in Mitochondrial Carrier family proteins. We demonstrate how charged or polar residues located in the middle of transmembrane helices can interfere with their identification by membrane topology tools resulting in missed helices in the prediction. Comparison of AAC in the human proteins with that in 235 secondary transport proteins from Escherichia coli revealed similar overall trends along with differences in average contents for some individual amino acids and groups of similar amino acids that are presumed to result from a greater number of functions and complexity in the higher organism.     

l- andd-alanine transport in brush border membrane vesicles from lepidopteran midgut: Evidence for two transport systems

Summary In brush border membrane vesicles from the midgut ofPhilosamia cynthia larvae (Lepidoptera) thel- andd-alanine uptake is dependent on a potassium gradient and on transmembrane electrical potential difference. Each isomer inhibits the uptake of the other form: inhibition ofl-alanine uptake byd-alanine is competitive, whereas inhibition ofd-alanine uptake byl-alanine is noncompetitive. Transstimulation experiments as well as the different pattern of specificity to cations suggest the existence of two transport systems. Kinetic parameters for the two transporters have been calculated both when K_out>K_in and K_out=K_in.d-alanine is actively transported also by the whole midgut, but it is not metabolized by the intestinal tissue.     

Chiral calix[4]arenes bearing amino alcohol functionality as membrane carriers for transport of chiral amino acid methylesters and mandelic acid

Novel chiral calix[4]arene derivatives bearing amino alcohol moieties at the lower rim have been synthesized from the reaction of p-tert-butylcalix[4]arene diester with various amino alcohols. The transport of amino acid esters (phenylglycine, phenylalanine, and tryptophan methyl esters hydrochloride) and mandelic acid were studied through chloroform bulk liquid membrane system using chiral calix[4]arenes 15-20. All these receptors have been found to act as carriers for transport of aromatic amino acid methylesters and mandelic acid from the aqueous source phase to the aqueous receiving phase. The influence of calixarene and guest structures upon transport through liquid membrane is discussed.     

Mechanisms and specificity of amino acid transport in Taenia crassiceps larvae (Cestoda)

The absorption of lysine, arginine, phenylalanine and methionine by Taenia crassiceps larvae is linear with respect to time for at least 2 min. Arginine uptake occurs by a mediated system and diffusion, and arginine, lysine and ornithine (in order of decreasing affinity) are completely competitive inhibitors of arginine uptake. The basic amino acid transport system has a higher affinity for l-amino acids than d-amino acids, and blocking the α-amino group of an amino acid destroys its inhibitory action. Phenylalanine uptake by T. crassiceps larvae is inhibited in a completely competitive fashion by serine, leucine, alanine, methionine, histidine, phenylalanine, tyrosine and tryptophan (in order of increasing affinity). Methionine apparently binds non-productively to the phenylalanine (aromatic amino acid-preferring) transport system. l-methionine uptake by larvae is inhibited more by d-alanine and d-valine than by their respective l-isomers, while d- and l-methionine inhibit l-methionine uptake equally well. The presence of an unsubstituted α-amino group is essential for an inhibitor to have a high affinity for the methionine transport system. Uptake of arginine, phenylalanine and methionine is Na⁺-insensitive, and both phenylalanine and methionine are accumulated by larvae against a concentration difference in the presence or absence of Na⁺. Arginine accumulation is precluded by its rapid metabolism to proline, ornithine and an unidentified compound.     

Anilinonaphthalene sulfonate fluorescence and amino acid transport in yeast

Summary Fluorescence of 1-anilinonaphthalene-8-sulfonate in yeast membranes appears to be caused predominantly by binding to lipids (ANS_proteinANS_lipid120) as indicated by the fluorescence lifetime, degree of polarization, and excitation spectra. It was insensitive to short-circuiting the membrane potential. Fluorescence intensity increased as cells (especially after pretreatment with energy donors such as glucose) were exposed to some amino acids, in particular, aspartic and glutamic acids. The character of fluorescence shifted to that of protein-bound ANS, suggesting an exposure of new protein sites accessible to the probe. This shift could be prevented by inhibitors of energy transduction as well as of transport. TheK _1/2 of the shift was at 2.5mm aspartic acid.     

Characterization of amino-acid transport in Ricinus communis roots using isolated membrane vesicles

The mechanism and specificity of amino-acid transport at the plasma membrane of Ricinus communis L. roots was investigated using membrane vesicles isolated by phase partitioning. The transport of glutamine, isoleucine, glutamic acid and aspartic acid was driven by both a pH gradient and a membrane potential (internally alkaline and negative), created artificially across the plasma membrane. This is consistent with transport via a proton symport. In contrast, the transport of the basic amino acids, lysine and arginine, was driven by a negative internal membrane potential but not by a pH gradient, suggesting that these amino acids may be taken up via a voltage-driven uniport. The energized uptake of all of the amino acids tested showed a saturable phase, consistent with carrier-mediated transport. In addition, the membrane-potential-driven transport of all the amino acids was greater at pH 5.5 than at pH 7.5, which suggests that there could be a direct pH effect on the carrier. Several amino-acid carriers could be resolved, based on competition studies: a carrier with a high affinity for a range of neutral amino acids (apart from asparagine) but with a low affinity for basic and acidic amino acids; a carrier which has a high affinity for a range of neutral amino acids except isoleucine and valine, but with a low affinity for basic and acidic amino acids; and a carrier which has a higher affinity for basic and some neutral amino acids but has a lower affinity for acidic amino acids. The existence of a separate carrier for acidic amino acids is discussed.Abbreviations PM plasma membrane - TPP⁺ tetraphenylphosphonium ion - pH pH gradient - membrane potential This work was supported by the Agricultural and Food Research Council and The Royal Society. We would like to thank Mrs. Sue Nelson for help with some of the membrane preparations.     

Genetic control of amino acid transport in sheep erythrocytes

An inherited amino acid transport deficiency results in low concentrations of glutathione (GSH) in the erythrocytes of certain sheep. Earlier studies based on phenotyping according to GSH concentrations indicated that the gene Tr ^H, which controls normal levels of GSH, behaves as if dominant or incompletely dominant to the allele Tr ^h, which controls the GSH deficiency. The present paper shows that when sheep are classified according to amino acid transport activity, the Tr ^H gene behaves as if codominant to Tr ^h. Erythrocytes from sheep homozygous for the Tr ^H gene exhibit rapid saturable l-alanine influx (apparent K _m ,21.6mm; V _max, 22.4 mmol/liter cells/hr). Cells from sheep homozygous for the Tr ^h gene exhibit slow nonsaturable l-alanine uptake (0.55 mmol/liter cells/hr at 50mm extracellular l-alanine). Cells from heterozygous sheep show saturable l-alanine uptake with a diminished V _max (apparent K _m, 19.1mm; V _max, 12.7 mmol/liter cells/hr). These erythrocytes have a significantly lower GSH concentration than cells from Tr ^H, Tr^H sheep but similar intracellular levels of dibasic amino acids.The authors are grateful to the M.R.C. for a Project Grant.     

Membrane vesicles from brown adipose tissue: A tool for the study of amino acid transport. The case of L-alanine

A density gradient method is used to isolate membrane vesicles from brown adipose tissue. These respond to changes in osmolarity and show the classical overshoot pattern when L-alanine uptake is assayed. Transport is shown to be effected by two components: a linear (K_d=0.498 min⁻¹) and Na⁺-dependent saturable component (K_m=2.3 mM) and a V_max=19.9 pmol/μg protein·min). This pattern is similar to that shown by cells isolated from brown adipose tissue.     

Evidence for a regulatory element controlling amino aced transport system L in Chinese hamster ovary cells

We have used the technique of somatic cell hybridization to study the regulation of the neutral amino acid transport system L in Chinese hamster ovary (CHO) cells. The cell line CHO–;tsO25C1 has a temperature-sinsitive mutationin leucyl-tRNA synthetase. At the nonpermissive temperature of 39^oC, CHO–tsO25C1 cells are unable to charge leucyl-tRNA and behave as though starved for leucine by increasing their system L transport activity two- to fourfold. From the temperature-sensitive cell line, we have isolated a regulatory mutant cell, CHO–C11B6, that has constitutively elevated system L transport activity. The CHO–C11B6 cell line retains the temperature-sensitive leucyl-tRNA synthetase mutation, but growth of this cell line is temperature resistant because its increased system L transport activity leads of increased intracellular leucine levels, which compensate for the defective. Hybrid cells formed by fusion of the temperature-sensitive CHO–;tsO25C1 cells the temperature-resistant CHO–C11B6 cells show temperature-sensitive growth and temperature-dependent regulation of leucine transport activity. These data suggest that the system L activity of CHO cells is regulated by a dominant-acting element that is defective or absent in the regulatory mutant CHO–C11B6 cell line.     

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.     

Cationic and anionic amino acid transport studies in rat red blood cells

The transport of L-proline, L-lysine and L-glutamate in rat red blood cells has been studied. L-proline and L-lysine uptake were Na⁺-independent. When the concentration dependence was studied both showed a non-saturable uptake assimilable to a difussion-like process, with high Kd values (0.718 and 0.191 min^–1 for L-proline and L-lysine respectively). Rat red blood cells showed high impermeability to L-glutamate. No sodium dependence was observed and the Kd value was low (0.067 min^–1). Our results show firstly, that rat red blood cells do not have amino acid transport systems for anionic and cationic amino acids and secondly that erythrocytes show no sodium-dependent L-proline transport, and that these cells are very permeable to this amino acid.Abbreviations MeAIB methyl aminoisobutyric acid     

Regulation of amino acid transport in growing cells of Streptomyces hydrogenans

(1) The active uptake of different amino acids by growing cells of Streptomyces hydrogenans was shown to be correlated with the physiological age of the cells. During the lag phase of growth the transport capacity increased and attained its highest level when the growth rate was maximum. During further growth the transport capacity declined progressively. The lowest transport activity was observed when the culture shifted into the stationary growth phase. (2) Such modulation of transport capacity was independent on the presence or absence of amino acids in the growth medium of the cells. (3) The size and the composition of the pool of free intracellular amino acids was also undergoing substantial variations during the growth cycle of the culture. In the lag phase, the levels of all amino acids decreased markedly and attained their lowest values at the end of this phase. During further growth the pool size was slowly replenished. (4) Removal of the pool resulted in a considerable gain of transport capacity. Therefore, it was concluded that active amino acid transport in growing Streptomyces hydrogenans is under feedback control by intracellular amino acids. (5) Quantitatively, the modulation of the pool size could not fully account for the variation of the transport capacity. Since a pool-independent stimulation of transport was found to be correlated with the increase of the growth rate of the cells, the possibility is discussed that the stimulation of transport is either due to increased levels of distinct RNA species, which might provide positive feedback signals for transport, or by increased rates of de novo synthesis of transport limiting proteins.List of Abbreviations AIB 2-aminoisobutyric acid - CM complete medium - MM mineral medium     

Regulation of the plasma amino acid profile by leucine via the system L amino acid transporter

Plasma concentrations of amino acids reflect the intracellular amino acid pool in mammals. However, the regulatory mechanism requires clarification. In this study, we examined the effect of leucine administration on plasma amino acid profiles in mice with and without the treatment of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) or rapamycin as an inhibitor of system L or mammalian target of rapamycin complex 1, respectively. The elevation of plasma leucine concentration after leucine administration was associated with a significant decrease in the plasma concentrations of isoleucine, valine, methionine, phenylalanine, and tyrosine; BCH treatment almost completely blocked the leucine-induced decrease in plasma amino acid concentrations. Rapamycin treatment had much less effects on the actions of leucine than BCH treatment. These results suggest that leucine regulates the plasma concentrations of branched-chain amino acids, methionine, phenylalanine, and tyrosine, and that system L amino acid transporters are involved in the leucine action.     

Sheep twin chimaeras with admixtures of red cell amino acid and potassium transport phenotypes

A pair of sheep twins each had two populations of red cells. Population 1 was positive for antigens Aa, Ma and Mb, was low-potassium type, possessed an amino acid transport system and was lysine-negative phenotype. Population 2 was negative for antigens Aa, and Mb, was high-potassium type, lacked the amino acid transport system and was lysine-positive phenotype. Population 2 disappeared from both sheep over a period of 8 years.     

Energy sources for amino acid transport in animal cells

The existence of an electrogenic Na⁺ pump in Ehrlich cells which substantially contributes to the membrane potential, previously derived from the distribution of the lipid soluble cation tetraphenylphosphonium (TPP⁺), could be confirmed by an independent method based on the quenching of fluorescence of a cyanine dye derivative, after the mitochondrial respiration had been suppressed by appropriate inhibitors. The mitochondrial membrane potential, by adding to the overall potential as measured in this way is likely to cause an overestimation of the membrane potential difference (p.d.). But since this error tends to diminish with increasing pump activity, the true p.d. of the plasma membrane should easily account for the driving force to drive the active accumulation of amino acids in the absence of an adequate Na⁺ concentration gradient. Accordingly, the F₂-aminoisobutyric acid (AIB) uptake rises linearly with the distribution of TPP⁺ at constant Na⁺ concentrations, suggesting that each responds directly to membrane potential. There is evidence that the electrogenic (free) movement of Cl^? is slow, at least at normal p.d., whereas a major part of the Cl^? movement across the cellular membrane appears to occur by an electrically silent Cl^?-base exchange mechanism. By such a mode Cl^?, together with an almost stoichiometric amount of K⁺, may under certain conditions move into the cell against a high adverse electrical potential difference. This “paradoxical” movement of K⁺Cl^? contributing to the deviation of the Cl^? distribution from the electrochemical equilibrium distribution, is not completely understood. It is insensitive towards ouabain but can almost specifically be inhibited by furosemide. As a likely explanation a H⁺–K⁺ exchange pump was previously offered, even though unequivocal evidence of such a pump is so far lacking. According to available evidence the electrogenic movement of free Cl^? is too small, at least at normal orientation of the p.d., to significantly shunt the electrogenic pump potential so that the establishment of such a potential is plausible. The evidence presented is considered strong in favor of the gradient hypothesis since even in the absence of an adequate Na⁺ concentration gradient, the electrogenic Na⁺ pump will contribute sufficient extra driving force to actively transport amino acid into the cells.     

ELF magnetic fields increase amino acid uptake into Vicia faba L. roots and alter ion movement across the plasma membrane

Vicia faba seedlings, subjected to a 10 microT 50 Hz square wave magnetic field for 40 min together with a radioactive pulse, showed a marked increase in amino acid uptake into intact roots. A more modest increase was observed with a 100 microT 50 Hz square wave. An increase in media conductivity at low field intensities from 10 microT 50 Hz square wave, 100 microT 50 Hz sine wave, and 100 microT 60 Hz square wave fields, indicated an alteration in the movement of ions across the plasma membrane, most likely due to an increase in net outflow of ions from the root cells. Similarly, marked elevation in media pH, indicating increased alkalinity, was observed at 10 and 100 microT for both square and sine waves at both 50 and 60 Hz. Our data would indicate that low magnetic field intensities of 10 and 100 microT at 50 or 60 Hz can alter membrane transport processes in root tips.     

Absence of a role of gamma-glutamyl transpeptidase in the transport of amino acids by rat renal brushborder membrane vesicles

Summary The role of the enzyme, gamma-glutamyl transpeptidase on the uptake of amino acids by the brushborder membrane of the rat proximal tubule was examined by inhibiting it with AT-125 (l-[S, 5S]--amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). AT-125 inhibited 98% of the activity of gamma-glutamyl transpeptidase when incubated for 20 min at 37°C with rat brushborder membrane vesicles. AT-125 given to ratsin vivo inhibited 90% of the activity of gamma-glutamyl transpeptidase in subsequently isolated brushborder membrane vesicles from these animals. AT-125 inhibition of gamma-glutamyl transpeptidase bothin vivo andin vitro had no effect on the brushborder membrane uptake of cystine. Similarly, there was no effect of gamma-glutamyl transpeptidase inhibition by AT-125 on glutamine, proline, glycine, methionine, leucine or lysine uptake by brushborder membrane vesicles. Furthermore, the uptake of cystine by isolated rat renal cortical tubule fragments, in which the complete gamma-glutamyl cycle is present, was unaffected by AT-125 inhibition of gamma-glutamyl transpeptidase. Therefore, in the two model systems studied, gamma-glutamyl transpeptidase did not appear to play a role in the transport of amino acids by the renal brushborder membrane.