Separate and shared lysosomal transport of branched and aromatic dipolar amino acids

Transport systems analogous to the T and L carriers for aromatic and bulky dipolar amino acids in plasma membranes have been characterized in the membranes of intact lysosomes isolated from human fetal skin fibroblasts. While system L appears ubiquitous in plasma membranes, system T has previously been discriminated only in the plasmalemma of human red blood cells and freshly isolated rat hepatocytes. Our findings with the lysosomal systems, provisionally designated t and l, reveal both shared and dissimilar properties with the plasma membrane systems. These properties include a lack of dependency on extralysosomal Na+, differential sensitivities to the classical system L analog, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), and the system T analog, D-tryptophan, as well as susceptibility to thiol modification at the membrane by reactivity with N-ethylmaleimide. A transport system in lysosomes from the FRTL-5 rat thyroid cell line has been described by Bernar et al. ((1986) J. Biol. Chem. 261, 17107-17112) resembles a composite of both carrier systems reported in this work.     

Phytomonas: transport of amino acids, hexoses and polyamines

Phytomonas cells (Phytomonas Jma) isolated from the latex of Jatropha macrantha were assayed for amino acid, hexose and polyamine transport. Results showed high transport rates for glucose and fructose (193 and 128 pmol min(-1) 10(-7) cells, respectively) and lower, but significant rates, for proline, arginine, cysteine and glutamate (between 1.7 and 5.8 pmol min(-1) 10(-7) cells). Minor transport activities were observed for serine, glycine and aspartate (<1 pmol min(-1) 10(-7) cells). Amino acid transport processes do not seem to be regulated by starvation or during the growth phases. Polyamine transport was also evaluated showing a clear preference for spermidine over putrescine (3.4 and 0.4 pmol min(-1) 10(-7) cells, respectively). This work represents the first report on metabolite transport in phytomonads.     

Erythrocytes participate significantly in blood transport of amino acids during the post absorptive state in normal humans

To investigate the participation of erythrocytes in the blood transport of amino acids during the course of intestinal absorption in humans, erythrocyte and plasma amino-acid concentrations were determined following ingestion of an oral load of amino acids. In addition to baseline plasma and erythrocyte amino acid concentrations in 18 subjects, plasma and erythrocyte amino acids kinetics during the 125 min following an oral amino acid load were further determined in 9 of the 18 subjects. The results showed that human erythrocytes contained most amino acids at similar or higher concentrations than plasma. Furthermore, the correlations observed between plasma and erythrocyte contents clearly indicated that erythrocytes were involved in the transport of amino acids by the blood. For some amino acids erythrocyte transport sometimes exceeded that of plasma. Significant correlation coefficients showed that strong plasma-erythrocyte relationships existed for alanine, valine, methionine, isoleucine, leucine, phenylalanine, and ornithine. In conclusion, our data supported the hypothesis that both blood compartments, plasma and erythrocytes, are involved significantly in the blood transport of amino acids in humans during the postabsorptive state. Accepted: 24 June 1998     

Synergism between different transport systems stimulates the uptake of neutral amino acids by isolated brain microvessels

Summary The polar long-chain amino acids glutamine and methionine can be transported across the endothelial cells of brain microvessels either by an L-system which operates by a facilitated diffusion, exchanging mechanism, or by a concentrating, energy-dependent A-system. The presence of glutamine and/or of methionine can induce a synergism between the two transport systems which results, by a transstimulation mechanism, in a net increased uptake of neutral hydrophobic aminoacids. The methionine analog S-methylthiocysteine, which is the mixed disulfide resulting from the combination of methanethiol with cysteine, behaves similarly to methionine in stimulating the uptake of neutral hydrophobic amino acids. The same transstimulating effect can even be obtained in collagenase-treated, A-system-deprived microvessels by inducing the direct formation of S-methylthiocysteine within the cytoplasmic compartment of the endothelial cells.Abbreviations MeAIB -methylaminoisobutyric acid - BCH DL--aminobicyclo-2,2,1-heptanecarboxylic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid     

The effect of altered ergosterol content on the transport of various amino acids in Candida albicans

Candida albicans cells have low levels of ergosterol when grown in ascorbic acid-supplemented media. When cells are grown in hydroquinone-supplemented media, the ergosterol levels became higher as compared to normal cells. The uptake of lysine, glycine, glutamic acid, proline, methionine and serine is reduced in hydroquinone-supplemented cells. In contrast to hydroquinone-supplemented cells, the rate and level of accumulation of these amino acids are higher in ascorbic acid-supplemented cells. Nystatin-resistant isolates of C. albicans with low ergosterol contents also exhibit an increased rate and level of accumulation of these amino acids. The uptake of phenylalanine and leucine remained unaffected by such a change in ergosterol levels brought about by different supplementation of the media. The results demonstrate a correlation between ergosterol levels and amino acids uptake. Contrary to various reports, the rate of K⁺ efflux does not seem to correlate with the amino acid uptake in C. albicans cells.     

The formation of vesicles retaining sodium-dependent transport systems for amino acids from protein-depleted membranes of pigeon erythrocytes

The process of the formation of vesicles from pigeon erythrocyte membranes was studied. Mildly alkaline solutions of low ionic strength, which reduce human erythrocyte membranes to small vesicles depleted of spectrin and other proteins, have no such effect on pigeon erythrocyte ghosts. A distinct phase of removal of membrane proteins, including spectrin, began to occur only when pigeon erythrocyte membranes were exposed to 0.2 mM EDTA adjusted to pH values above 10.2. Vesicles which demonstrated Na⁺-dependent amino acid transport were generated between the pH values 10.8 and 11.4. The results show that peripheral proteins, notably spectrin, maintain the integrity of the pigeon erythrocyte ghost. The interaction of these proteins with the membrane is rather different from that well studied in the human erythrocyte ghost and the possible significance of this for the pigeon erythrocyte is discussed.     

Characteristics of the active transport of peptides and amino acids by germinating barley embryos

Use of two different assays involving either radioactively labelled substrates or a fluorescent-labelling procedure, gave good agreement for the rates of transport of peptides and amino acids into the scutellum of germinating grains of barley (Hordeum vulgare cv. Maris Otter, Winter). However, evidence was obtained for the enzymic decarboxylation of transpored substrate, which can cause underestimates of transport rates when using radioactively labelled substrates. The peptide Gly-Phe, was shown to be rapidly hydrolysed after uptake, and autoradiography of transported Gly-[U-¹⁴C]Phe indicated a rapid distribution of tracer, i.e. [U-¹⁴C] phenylalanine into the epithelium and sub-epithelial layers of the scutellum. The developmental patterns of transport activity indicate that peptide transport is more important nutritionally during the early stages of germination (1–3 d) whereas amino acids become relatively more important later (4–6 d). A range of amino acids is shown to be actively transported and several compete for uptake. At physiological concentrations, e.g. 2mM, transport of peptides and amino acids is inhibited about 80% by protonophore uncouplers, but at higher concentrations (10–100 mM) passive uptake predominates.Abbreviations Gly glycine - Leu leucine - Phe phenylalanine - Pro proline     

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     

Trypanosoma gambiense: membrane transport of amino acids.

Trypanosoma gambiense absorbed ¹⁴C-labeled lysine, arginine, glutamate, phenylalanine, methionine, threonine, glycine, and alanine by mediated transport systems. The interactions of these compounds as inhibitors or stimulators formed complex patterns of uptake which suggested the presence of five binding and/or transport loci: Locus A bound glutamate, arginine, and lysine, and the binding of glutamate or arginine stimulated the transport of lysine. Locus B transported threonine, glycine, and alanine and appeared to be partially sensitive to ouabain and Na⁺. Locus C transported glutamate, locus D transported phenylalanine and methionine, and locus E transported lysine and arginine.     

Driving forces and current-voltage characteristics of amino acid transport in Riccia fluitans

The complete steady-state I–V relationship of α-aminoisobutyric acid transport across the plasmalemma of rhizoid cells from Riccia fluitans has been measured and analysed with special emphasis on α-aminoisobutyric acid equilibrium and saturation conditions. (A) The electrical data show that: (1) the amino acid-induced electrical current saturates after the addition of the amino acid, regardless of the concentration; (2) a steady state is reached 1–2 h after incubation in α-aminoisobutyric acid, but after less that 5 min in the presence of 1 mM CN⁻; (3) the steady-state I–V characteristic of α-aminoisobutyric acid transport is a sigmoid curve and fairly symmetric in current with respect to the voltage axis; and (4) the equilibrium potential is clearly a function of the amino acid accumulation ratio. It is suggested that the sigmoid curve represents the characteristic of carrier-mediated α-aminoisobutyric acid transport with a voltage-insensitive step, possibly the translocation of the unloaded carrier, rate-limiting. Since under normal conditions the voltage-sensitive rate constant k_oi is much greater than k_io, it is further suggested that the energy to drive this system is put into the transfer of positive charge from outside to the cytoplasm. (B) Accumulation ratios have been determined by inspection of current-voltage data, and additionally by compartmental analysis on green thalli from Riccia fluitans. Both methods give ratios far too low compared with the thermodynamically possible accumulation of about 10⁴. It is suggested that substantial leakages via different non-electrical pathways prevent equilibrium at steady state, and it is concluded that in such leaky systems the thermodynamic equilibrium condition is not suitable for estimating stoichiometries.     

Comparative analysis of amino acid metabolism and transport in CHO variants with different levels of productivity

Chinese hamster ovary (CHO) cells are widely used for the production of biopharmaceuticals; however, our understanding of several physiological elements that contribute to productivity is limited. One of these is amino acid transport and how its limitation and/or regulation might affect productivity. To further our understanding, we have examined the expression of 40 mammalian amino acid transporter genes during batch cultures of three CHO cell lines: a non-producer and two antibody-producing cell lines with different levels of productivity. In parallel, extracellular and intracellular levels of amino acids were quantified. The aim was to identify differences in gene regulation between cell lines and within culture. Our results show that three transporters associated with transport of taurine and β-alanine, acidic amino acids and branched chain amino acids, are highly upregulated in both antibody-producing cell lines but not in the non-producer. Additionally, genes associated with the transport of amino acids related to the glutathione pathway (alanine, cysteine, cystine, glycine, glutamate) were found to be highly upregulated during the stationary phase of cell culture, correlating well with literature data on the importance of the pathway. Our analysis highlights potential markers for cell line selection and targets for process optimization.     

Solute transport across the tonoplast of barley mesophyll vacuoles: Mg2+ determines the specificity,and ATP and lipophilic amino acids the activity of the amino acid carrier

After Stimulation with ATP and in the absence of divalent cations, isolated barley mesophyll vacuoles exhibited massive solute fluxes across the tonoplast, measured either as efflux of endogenous solutes or as uptake of radioactive-labeled compounds. Transported solutes were ions (particularly K⁺, NO ₃ ^– , Cl^–) and amino acids (for example, ala, arg, asp, gln, leu, met). Addition of Mg²⁺in excess of added ATP inhibited fluxes of inorganic ions and of positively charged amino acids, but not, or to a smaller extent, those of neutral amino acids. Thus, Mg²⁺ increased the specificity of the carrier for amino acids such as alanine and glutamine. All ATP-stimulated transport processes were sensitive towards inhibition by lipophilic amino acids, for example by leucine and phenylalanine. After stimulation with sulfhydryl reagents, the inhibitory properties of Mg²⁺ and lipophilic amino acids were lost. These data concur with the hypothesis of a single transporter which exhibits a channel-like structure with a low degree of substrate selectivity in the absence of Mg²⁺, and which functions as a neutral amino acid carrier in the presence of Mg²⁺.We are grateful to Frau Claudia Dürr for excellent technical assistance. The work was supported by the Deutsche Forschungsgemeinschaft within the Sonderforschungsbereich 176 of the Bayerische-Julius-Maximilians-Universität Würzburg.     

Transport of neutral amino acids by human erythrocytes

The transport of several neutral amino acids by human erythrocytes in vitro was studied. The measurements made included steady-state distributions, kinetics of initial rates of uptake, effects of monovalent cations and anions, general mutual inhibitory interactions, kinetics of inhibitions, effluxes, ability to produce accelerative exchange diffusion, and the inhibitory action of the thiol reagent N-ethylmaleimide. The results are interpreted as showing that the human erythrocyte membrane possesses several distinct transport systems for these amino acids, including one Na⁺-dependent system and one dependent on both Na⁺ and a suitable anion, that are qualitatively similar to those systems previously described in pigeon erythrocytes and mammalian reticulocytes. Quantitatively, however, the systems differ among the different kinds of red cell and a major difference lies in their abilities to produce accelerative exchange diffusion.     

Inhibition of 3,4-dihydroxy-l-phenylalanine decarboxylase in rat striatal synaptosomes by amino acids interacting with substrate transport

Dopamine synthesis from 3,4-dihydroxy-l-phenylalanine in rat striatal synaptosomes was inhibited by a number of amino acids with aromatic or large aliphatic side chains. Inhibition was not seen when aromatic amino acid decarboxylase activity was measured in disrupted synaptosomes. Similarly, inhibition of dopamine synthesis from tyrosine was seen in the presence of leucine. The inhibition most likely results from interactions of the amino acids with substrate transport across the synaptosome plasma membrane, rather than directly with the catalytic enzymes. The kinetic data obtained are used to infer information about the relevant transport process; they suggest the potential importance of amino acid efflux as a regulatory step.     

Interaction of amino acids with glycyl-glycine transport in the mammalian intestine

In order to investigate a possible interaction between free amino acids and dipeptides during their mucosal uptake in man and monkey, perfusion studiesin vivo and uptake studiesin vitro using labelled and non-labelled dipeptides and amino acids have been carried out. In contrast to the observations of other workers, inhibition of glycyl-glycine uptake was observed with free leucine and methioninc but not with glycine, proline, hydroxyproline or alanine. Leucine and methionine caused inhibition of cytosol glycyl-glycine hydrolase activity, while glycine had no effect. The dipeptide uptake and dipeptide hydrolysis by cytosol enzyme was competitively inhibited by leucine. Although brush border glycyl-glycine hydrolase was also inhibited by leucine, the inhibition was noncompetitive. These data indicate that a few free amino acids can interact with dipeptides during uptake. This interaction might occur either at the transport step or at the stage of intracellular dipeptide hydrolysis. The work reported here was carried out at Wellcome Research Unit, Christian Medical College and Hospital, Vellore 632 004.     

Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

The effect of heat shock on amino acid transport and cell volume in 3T3 cells

Summary. In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44°C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response. Received June 30, 1999 Accepted July 27, 2000     

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.     

Epidermal growth factor (EGF) increases the renal amino acid transport capacity in amino acid loaded rats

Summary In anaesthetized adult female rats, the influence of epidermal growth factor (EGF) on renal amino acid handling was investigated in glutamine, arginine (both 50 mg/100 g b. wt. per hour), or alanine (90 mg/ 100 g b. wt. per hour) loaded animals. Continuous infusions of the three amino acids were followed by an increase in the fractional excretion (FE) of the administered amino acids as well as of the other endogenous amino acids. Under load conditions (alanine, arginine or glutamine), EGF pretreatment (8g/100g b. wt. subcutaneously for 8 days, twice daily 8 a.m. and 4 p.m.) was followed by a stimulation of renal amino acid reabsorption. The increase in the fractional excretion of the administered amino acids was significantly lower than in non-EGF-treated rats. These changes in amino acid transport were connected with a significant reduction of GFR after EGF pretreatment (0.96 ± 0.10 vs. 0.62 ± 0.07 ml/min X 100 g b. wt.) and a distinct increase in sodium excretion (2.98 ± 0.55 vs. 4.97 ± 0.71val/100 g b. wt. X 20 min). After loading with p-aminohippurate (PAH; 200mg/100g b. wt.), PAH excretion in EGF rats was increased by about 20%, whereas urinary protein excretion was lower in EGF pretreated rats (control: 0.45 ± 0.04 vs. EGF: 0.18 ± 0.03 mg/ 100 g b. wt. X 20 min). The PAH load reduced amino acid reabsorption as a sign of overloading of renal tubular transport capacity, but in EGF pretreated animals the amino acid excretion was only slightly increased under these conditions. Furthermore, EGF pretreatment depressed normal kidney weight gain significantly (874 ± 18 vs. 775 ± 32mg/100g b. wt.). EGF can improve the renal tubular transport capacity, but, compared to well-known stimulators of renal transport like dexamethasone or tri-iodothyronine, its effect is only of a moderate degree.