Effect of spermine on the uptake of amino acids in Micrococcus lysodeikticus

Spermine inhibited the transport of neutral aliphatic amino acids (valine, leucine, isoleucine, alanine, and glycine) into cells of Micrococcus lysodeikticus. On the other hand, spermine did not affect the uptake of basic (arginine and histidine), acidic (glutamic acid), or aromatic (phenylalanine and tyrosine) amino acids. Inhibition of uptake of the neutral amino acids by spermine is apparently of a noncompetitive nature; the V(max) decreased, whereas the apparent K(m) remained unaltered. The inhibition is most likely due to a specific binding of spermine to the carrier(s) of these amino acids. Related polyamines, spermidine and cadaverine, also caused inhibition of valine uptake, though to a lesser extent; spermidine was less active than spermine, and cadaverine showed the weakest effect of all. Valine, leucine, and isoleucine were transported into M. lysodeikticus cells by a common carrier as evidenced from competition experiments. The uptake of these amino acids is an active process; it was temperature-dependent and inhibited by azide (10(-1)m to 2.5 x 10(-2)m) and dinitrophenol (10(-3)m). The intracellular concentration of valine was 100-fold higher than in the medium.     

Inhibition by Peptides of Amino Acid Uptake by Bacterial Populations in Natural Waters: Implications for the Regulation of Amino Acid Transport and Incorporation

To investigate the regulatory interactions of amino acid transport and incorporation, we determined the effects of dipeptides on amino acid uptake by bacteria in an estuary and a freshwater lake. Dipeptides noncompetitively inhibited net transport and incorporation of amino acids into macromolecules but had no effect on the ratio of respiration to incorporation. Nearly maximum inhibition occurred at peptide concentrations of <10 nM. In contrast, the initial uptake rate of glycyl-[¹⁴C]phenylalanine was not affected by glycine or phenylalanine. Net amino acid transport appeared to be inhibited by the increased flux into the intracellular pools, whereas the incorporation of labeled monomers into macromolecules was isotopically diluted by the unlabeled amino acids resulting from intracellular hydrolysis of the dipeptide. Chloramphenicol, sodium azide, and dinitrophenol all inhibited the initial uptake rate of leucine and phenylalanine. These results suggest that in aquatic environments amino acids are taken up by active transport which is coupled closely to protein synthesis.     

Metabolic and secretory responses of parotid cells to cationic amino acids. Oxidation of the amino acids and interference with the oxidation of D-glucose or endogenous nutrients.

Cationic amino acids were recently found to stimulate amylase release from rat parotid cells. The possible relevance of their oxidative catabolism to such a secretory stimulation was investigated. D-Glucose, which was efficiently metabolized in parotid cells and which augmented O2 uptake above basal value, failed to affect basal or stimulated amylase release. L-Arginine, L-lysine and L-histidine failed to stimulate the oxidation of either exogenous D-[6-14C]glucose or endogenous nutrients in cells pre-labelled with [U-14C]palmitate or L-[U-14C]glutamine. The oxidation of L-[U-14C]arginine, L-[U-14C]ornithine, L-[U-14C]lysine and L-[U-14C]histidine, all tested at a 10 mM concentration, was much lower than that of D-[U-14C]glucose (5.6 mM). These findings argue against the view that the stimulation of amylase release by cationic amino acids would be related to their role as a source of energy in the parotid cells.     

EVIDENCE FOR SEPARATE SYSTEMS FOR THE TRANSPORT OF NEUTRAL AND BASIC AMINO ACIDS ACROSS THE BLOOD-BRAIN BARRIER

Abstract— The effects of high circulating concentrations of several amino acids on the free amino acids of rat brain were measured, to see whether or not the results followed any consistent pattern. High circulating concentrations of large, neutral amino acids (phenylalanine, valine or isoleucine) caused significantly decreased values only of other large, neutral amino acids in the brains. High circulating concentrations of the basic amino acids lysine or arginine caused significantly decreased values only of each other. The data suggest that there are separate systems for the transport of neutral and basic amino acids across the blood-brain barrier. The effects of valine and lysine on the uptake by brain and the con-vulsant action of allylglycine (a neutral amino acid) were consistent with the concept of separate systems for the transport of amino acids across the blood-brain barrier. Valine inhibited the uptake by brain and the convulsant action of allylglycine in mice, but lysine did not. The data suggest that allylglycine and valine are transported into the brain by a common mechanism that does not transport lysine.     

Accumulation of large neutral amino acids in the brain of sparse-fur mice at hyperammonemic state

Sparse-fur mice which are deficient in ornithine transcarbamylase, the second-step enzyme in the urea cycle, were examined for hyperammonemia and its relationship with encephalopathy. We compared amino acid concentrations in the serum and brain of spf mice with those of control mice. Unlike hepatic encephalopathy we could not find marked amino acid changes in the serum of spf mice besides low levels of citrulline and arginine. But in the brain of spf mice, glutamine was increased strikingly during hyperammonemia, and a concomitant accumulation of large neutral amino acids such as tyrosine, phenylalanine, methionine, and histidine was observed. The accumulation of these large neutral amino acids in the brain was not influenced by 24-hr fasting which caused increases in branched chain amino acids in the serum. From these results, we conclude that the accumulation of the large neutral amino acid in the brain of hyperammonemic state is caused by uptake of ammonia in the brain and the subsequent accumulation of glutamine, but is not influenced by a decreased ratio of branched chain amino acids to aromatic amino acids in the serum.     

Ornithine transport and exchange in Streptococcus lactis.

Resting cells of Streptococcus lactis 133 appeared to accumulate [14C]ornithine to a high concentration in the absence of an exogenous energy source. However, analysis of intracellular amino acid pool constituents and results of transport experiments revealed that the accumulation of ornithine represented a homoexchange between extracellular [14C]ornithine and unlabeled ornithine in the cell. The energy-independent exchange of ornithine was not inhibited by proton-conducting uncouplers or by metabolic inhibitors. Intracellular [14C]ornithine was retained by resting cells after suspension in a buffered medium. However, addition of unlabeled ornithine to the suspension elicited rapid exit of labeled amino acid. The initial rate of exit of [14C]ornithine was dependent on the concentration of unlabeled ornithine in the medium, but this accelerative exchange diffusion process caused no net loss of amino acid. By contrast, the presence of a fermentable energy source caused a rapid expulsion of and net decrease in the concentration of intracellular ornithine. Kinetic analyses of amino acid transport demonstrated competitive inhibition between lysine and ornithine, and data obtained by two-dimensional thin-layer chromatography established the heteroexchange of these basic amino acids. The effects of amino acids and of ornithine analogs on both entry and exit of [14C]ornithine have been examined. The data suggest that a common carrier mediates the entry and exchange of lysine, arginine, and ornithine in cells of S. lactis.     

AAP1 transports uncharged amino acids into roots of Arabidopsis

Amino acids are available to plants in some soils in significant amounts, and plants frequently make use of these nitrogen sources. The goal of this study was to identify transporters involved in the uptake of amino acids into root cells. Based on the fact that high concentrations of amino acids inhibit plant growth, we hypothesized that mutants tolerating toxic levels of amino acids might be deficient in the uptake of amino acids from the environment. To test this hypothesis, we employed a forward genetic screen for Arabidopsis thaliana mutants tolerating toxic concentrations of amino acids in the media. We identified an Arabidopsis mutant that is deficient in the amino acid permease 1 (AAP1, At1g58360) and resistant to 10 mm phenylalanine and a range of other amino acids. The transporter was localized to the plasma membrane of root epidermal cells, root hairs, and throughout the root tip of Arabidopsis. Feeding experiments with [(14)C]-labeled neutral, acidic and basic amino acids showed significantly reduced uptake of amino acids in the mutant, underscoring that increased tolerance of aap1 to high levels of amino acids is coupled with reduced uptake by the root. The growth and uptake studies identified glutamate, histidine and neutral amino acids, including phenylalanine, as physiological substrates for AAP1, whereas aspartate, lysine and arginine are not. We also demonstrate that AAP1 imports amino acids into root cells when these are supplied at ecologically relevant concentrations. Together, our data indicate an important role of AAP1 for efficient use of nitrogen sources present in the rhizosphere.     

Transport of basic amino acids in Riccia fluitans: Evidence for a second binding site

The transport of several amino acids with different side-chain characteristics has been investigated in the aquatic liverwort Riccia fluitans. i) The saturation of system I (neutral amino acids) by addition of excess -aminoisobutyric acid to the external medium completely eliminated the electrical effects which are usually set off by neutral amino acids. Under these conditions arginine and lysine significantly depolarized the plasmalemma. ii) L- and D-lysine/arginine were discriminated against in favour of the L-isomers. iii) Increasing the external proton concentration in the interval pH 9 to 4.5 stimulated plasmalemma depolarization, electrical net current, and uptake of [¹⁴C]-basic amino acids. iv) Uptake of [¹⁴C]-glutamic acid took place only at acidic pHs. v) [¹⁴C]-histidine uptake had an optimum between pH 6 and 5.5. vi) Overlapping of the transport of basic, neutral, and acidic amino acids was common. It is suggested that besides system I, a second system (II), specific for basic amino acids, exists in the plasmalemma of Riccia fluitans. It is concluded that the amino-acid molecule with an uncharged side chain is the substrate for system I, which also binds and transports the neutral species of acidic amino acids, whereas system II is specific for amino acids with a positively charged side chain. The possibility of system II being a proton cotransport is discussed.Abbreviation AiB -aminoisobutyric acid     

Rhamnogalacturonan-II--a biologically active fragment

Rhamnogalacturonan-II inhibited the uptake of [¹⁴C]leucine and,consequently, the incorporation of [¹⁴C]leucine into acid-precipitableproteins by suspension-cultured tomato cells. Fractionationof rhamnogalacturonan-II showed that the lower molecular componentswere the most effective. KDO and apiose, both constituents ofrhamnogalacturonan-II, also inhibited [¹⁴C]leucine incorporationweakly, suggesting that these sugar residues may be an integralrequirement for the biological activity of rhamnogalacturonan-II.The incorporation of [¹⁴C]glutamate and [¹⁴C]histidine, andto a lesser extent [¹⁴C]proline and [¹⁴C]arginine, was alsoinhibited by rhamnogalacturonan-II; the incorporation of [¹⁴C]tyrosineand [¹⁴C]phenylalanine was little affected. This suggests thatrhamnogalacturonan-II exerts its effect by acting on certainmembrane transport systems. Key words: Rhamnogalacturonan-II, inhibition, protein synthesis, amino acid incorporation     

Metabolism of L-(U-14C)valine, L-(U-14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated perfused lactating guinea-pig mammary gland.

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.     

Comparison of the inhibitory effects of diverse amino acids and amino acid analogs on 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity in isolated epidermal cells

At a concentration of 1.25 mM, 14 amino acids were capable of inhibiting the induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in isolated epidermal cells. The greatest percentages of inhibition of TPA-induced epidermal ornithine decarboxylase activity were as follows: cysteine, 98%; tryptophan, 74%; methionine, 64%; phenylalanine, 51%; glycine, 44%; asparagine, 43%; glutamic acid, 42%; leucine, 40%; and arginine, 39%. These amino acid treatments did not alter the time- and concentration-response curves for induction of ornithine decarboxylase activity by TPA. Moreover, there was no difference between the rates at which [3H]arginine, [3H]leucine, [3H]phenylalanine, [3H]methionine, [3H]tryptophan and [14C]cysteine were taken up by freshly isolated epidermal cells or incorporated into epidermal proteins. Arginine, phenylalanine and methionine inhibited the induction of ornithine decarboxylase activity by the tumor promoter to degrees comparable to those elicited by their analogs canavanine and homoarginine, beta-2-thienyl-DL-alanine, and ethionine, respectively. These amino acids and amino acid analogs did not alter the overall rate of protein synthesis. In contrast, both the amino acids and their analogs increased the rates of proteolysis in isolated epidermal cells, an effect which correlated well with the abilities of these different compounds to inhibit TPA-induced ornithine decarboxylase activity. Moreover, both methionine and phenylalanine decreased the half-life and increased the rate of heat denaturation of the TPA-induced enzyme, a result identical to that obtained after treatment with the analogs ethionine and beta-2-thienyl-DL-alanine, respectively. Taken together, these results suggest that millimolar concentrations of exogenous amino acids might induce the synthesis of abnormal proteins and nonfunctional enzymes. Therefore, it is speculated that the uptake of unbalanced amounts of amino acids into the epidermal target cells might alter the stability and the ultrastructure of the TPA-stimulated enzyme just as the amino acid analogs do.     

Cysteine and Cystine Transport at the Blood-Brain Barrier

Abstract: The nature of cysteine and cystine uptake from the cerebral capillary lumen was studied in the rat using the carotid injection technique. [³⁵S]-Cysteine uptake was readily inhibited by the synthetic amino acid 2-amino-bicyclo(2,2,1)heptane-2-carboxylic acid (BCH), the defining substrate for the leucine-preferring (L) system in the Ehrlich ascites cell. The addition of non-radioactive alanine or serine, representatives of the alanine, serine, and cysteine-preferring (ASC) system, produced no significant decrease in the uptake of cysteine after cysteine transport by the L system was blocked with BCH. This indicated that the major component of cysteine's transport from the brain capillary lumen was by the L system with no detectable uptake of cysteine by the ASC system. No carrier-mediated transport of cystine, the disulfide form of the amino acid, was detected, nor was there any inhibition by cystine of the transport of the neutral amino acid methionine or the basic amino acid arginine. These results suggest that the ASC system, if present, is not quantitatively important for the transport of neutral amino acids from the brain capillary lumen.     

The association of lesion-induced reductions in brain monoamines with alterations in striatal carbohydrate metabolism

Newly-weaned male guinea pigs were fed an ascorbic acid-deficient diet ad libitum and compared with control animals pair-fed an adequate diet for a similar duration. The ascorbic acid-deficient animals demonstrated prominent elevations in serum concentrations of tyrosine (+427%), phenylalanine (+36%) and arginine (+21 %) with concomitant depressions in levels of glycine (–57 %), histidine (–39 %), ethanolamine (–38%) and glutamic acid (–22 %). With few exceptions, the alterations in the liver amino acid profiles were in the same directions as those observed in the serum. The scorbutic brains showed 28–36 per cent of the retention of total ascorbic acid found in control animals and were characterized by marked elevation (+83%) in tyrosine content, hardly any alteration in phenylalanine (–9%), and depressed levels of histidine (–33 %), arginine (–25%), phosphoserine (–50%) and GABA (–12%). The implications of such abnormal changes in free amino acid patterns were evaluated in the light of the role of some of these amino acids as precursors for the synthesis of neuroregulatory substances. No difference was observed in the brain polysomal profiles as isolated from the two groups of animals. Incubation of polysomes from ascorbic acid-deficient brains with autologous pH 5 enzyme derived from cell sap not passed through Sephadex G-25 revealed low uptake of [¹⁴C]phenylalanine in comparison to that for a similar system from control animals. Use of pH 5 enzymes prepared from Sephadex-treated and dialysed cell saps eliminated the difference in specific activities of the two groups of ribosomes, an observation suggesting that ascorbic acid deficiency either intensified the activity of the inhibitory components or reduced the low molecular weight stimulatory substances present under normal conditions in the brain postmicrosomal fraction.     

Effect of phenylalanine on protein synthesis in the developing rat brain

1. Inhibition of the rate of incorporation of [(35)S]methionine into protein by phenylalanine was more effective in 18-day-old than in 8-day-old or adult rat brain. 2. Among the subcellular fractions incorporation of [(35)S]methionine into myelin proteins was most inhibited in 18-day-old rat brain. 3. Transport of [(35)S]methionine and [(14)C]leucine into the brain acid-soluble pool was significantly decreased in 18-day-old rats by phenylalanine (2mg/g body wt.). The decrease of the two amino acids in the acid-soluble pool equalled the inhibition of their rate of incorporation into the protein. 4. Under identical conditions, entry of [(14)C]glycine into the brain acid-soluble pool and incorporation into protein and uptake of [(14)C]acetate into lipid was not affected by phenylalanine. 5. It is proposed that decreased myelin synthesis seen in hyperphenylalaninaemia or phenylketonuria may be due to alteration of the free amino acid pool in the brain during the vulnerable period of brain development. Amyelination may be one of many causes of mental retardation seen in phenylketonuria.     

Inhibition of Neutral Amino Acid Transport Across the Human Blood-Brain Barrier by Phenylalanine

Abstract: The delivery of large neutral amino acids (LNAAs) to brain across the blood-brain barrier (BBB) is mediated by the L-type neutral amino acid transporter present in the membranes of the brain capillary endothelial cell. In experimental animals, the L-system transporter is saturated under normal conditions, and therefore an elevation in the plasma concentration of one LNAA will reduce brain uptake of others. In this study, we used positron emission tomography (PET) to determine the effect of elevated plasma phenylalanine concentrations on the uptake of an artificial neutral amino acid, [¹¹C]-aminocyclohexanecarboxylate ([¹¹C]ACHC), in human brain. PET scans were performed on six normal male subjects after an overnight fast and again 60 min after oral administration of 100 mg/kg of phenylalanine. The plasma phenylalanine concentration increased by an average of 11-fold between the first and second scans. This increase produced a reduction in [¹¹C]ACHC uptake in all brain regions but not in scalp. The mean ± SD influx rate constant for whole brain decreased after phenylalanine ingestion from 0.036 ± 0.002 to 0.019 ± 0.004 ml/g/min. Kinetic analysis of the effect of plasma phenylalanine concentration on the rate of [¹¹C]ACHC uptake is compatible with a model of competitive inhibition so that large increases in the concentration of one LNAA in plasma will reduce the brain uptake of other LNAAs across the human BBB.     

Na+-Independent Transporters, LAT-2 and b0,+, Exchange L-DOPA with Neutral and Basic Amino Acids in Two Clonal Renal Cell Lines

The present study examined the functional characteristics of L-DOPA transporters in two functionally different clonal subpopulations of opossum kidney (OKLC and OKHC) cells. The uptake of L-DOPA was largely Na+-independent, though in OKHC cells a minor component (approximately 15%) required extracellular Na+. At least two Na+-independent transporters appear to be involved in L-DOPA uptake. One of these transporters has a broad specificity for small and large neutral amino acids, is stimulated by acid pH and inhibited by 2-aminobicyclo(2,2,l)-heptane-2-carboxylic acid (BCH; OKLC, Ki = 291 mM; OKHC, Ki = 380 mM). The other Na+-independent transporter binds neutral and basic amino acids and also recognizes the di-amino acid cystine. [14C]-L-DOPA efflux from OKLC and OKHC cells over 12 min corresponded to a small amount of intracellular [14C]-L-DOPA. L-Leucine, nonlabelled L-DOPA, BCH and L-arginine, stimulated the efflux of [14C]-L-DOPA in a Na+-independent manner. It is suggested that L-DOPA uses at least two major transporters, systems LAT-2 and b0,+. The transport of L-DOPA by LAT-2 corresponds to a Na+-independent transporter with a broad specificity for small and large neutral amino acids, stimulated by acid pH and inhibited by BCH. The transport of L-DOPA by system b0,+ is a Na+-independent transporter for neutral and basic amino acids that also recognizes cystine. LAT-2 was found equally important at the apical and basolateral membranes, whereas system b0,+ had a predominant distribution in apical membranes.     

Basic amino acid inhibition of cell division and macromolecular synthesis in Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae on poor nitrogen sources such as allantoin or proline was totally inhibited by addition of a non-degradable basic amino acid to the medium. Cells treated with lysine contained greatly reduced quantities of histidine and arginine. Conversely, lysine and histidine were severely reduced in arginase-deficient cells treated with arginine. When all three basic amino acids were present in the culture medium, growth was normal suggesting that synthesis of all three basic amino acids was decreased by an excess of any one of them. Inhibition of growth was accompanied by a fivefold increase in the observed ratio of budded to unbudded cells. These morphological changes suggested that DNA synthesis was inhibited. Consistent with this suggestion, addition of a basic amino acid to the culture medium substantially reduced the ability of the cells to incorporate [14C]uracil into alkali-resistant, trichloroacetic acid-precipitable material. RNA and protein synthesis, although decreased, were less sensitive to the effects of such additions.     

Tryptophan and phenylalanine transport in rat cerebral cortex slices as influenced by sodium ions

Tryptophan and phenylalanine transport in rat cerebral cortex slices was studied in sodium-free media and during influx and efflux of sodium ions. Choline as a substitute for sodium in incubation media increased efflux and decreased influx of tryptophan and phenylalanine. Exchange of intracellular [³H]tryptophan and [³H]phenylalanine with extracellular unlabeled histidine, phenylalanine, and tryptophan was sodium-independent. Efflux of sodium ions from the slices had no immediate effects on phenylalanine and tryptophan efflux, but influx decreased. Influx of sodium into the sodium-depleted slices provoked a transient increase in tryptophan and phenylalanine efflux and also enhanced influx. The results are interpreted to indicate that sodium ions may possibly affect the function of the primary transport sites for aromatic amino acids at cerebral membranes by controlling the orientation of their reactive sites towards the intracellular and extracellular sides, rather than by being directly involved in the binding of amino acids to the carriers.     

The effects of individual amino acids on the incorporation of labelled amino acids into proteins by brain homogenates

Abstract— The effects of phenylalanine and other amino acids on incorporation of several different ¹⁴C-labelled amino acids into cerebral protein were studied in brain homogenates. Excess of some amino acids had a varied effect with different ¹⁴C-labelled amino acids. Of the unlabelled-labelled amino acid combinations tested the maximal inhibition was obtained with the following: (1) phenylalanine, which inhibited the incorporation of [¹⁴C]tyrosine, and (2) leucine, which inhibited incorporation of [¹⁴C]isoleucine. In both cases the inhibition occurred principally in proteins that were recovered in the 800 g and 13,000 g sediments. Only a small degree of inhibition occurred in proteins that sedimented at 100,000 g, and no inhibition occurred in proteins of the 100,000 g supernatant.     

Effect of Aspartame-Derived Phenylalanine on Neutral Amino Acid Uptake in Human Brain: A Positron Emission Tomography Study

The possible effects of elevation of the plasma phenylalanine level secondary to the ingestion of aspartame on brain amino acid uptake in human subjects have been investigated by means of positron emission tomography (PET). 1-[11C]Aminocyclohexanecarboxylate [( 11C]ACHC) is a poorly metabolized synthetic amino acid that crosses the blood-brain barrier by the same carrier that transports naturally occurring large neutral amino acids. Quantitative test-retest PET studies were performed on 15 individuals. Seven received two identical baseline scans, whereas eight received a baseline scan followed by a scan performed approximately 40-45 min following ingestion of an orange-flavored beverage containing 34 mg/kg of body weight of the low-calorie sweetener aspartame, a dose equivalent to the amount in 5 L of diet soft drink consumed all at once by the study subjects, weighing an average of 76 kg. The 40-45-min interval was selected to maximize the detection of possible decreases in ACHC uptake resulting from increased competition for the carrier, because the plasma phenylalanine level is known to peak at this time. We observed an 11.5% decrease in the amino acid transport rate constant K1 and a smaller decrease in the tissue distribution volume of ACHC (6%). Under conditions of normal dietary use, aspartame is thus unlikely to cause changes in brain amino acid uptake that are measurable by PET.