Membrane-bound -glutamyl transpeptidase. Evidence that it is a component of the amino acid site' of certain neutral amino acid transport systems.
Explore the source record for details and available documents.
Acyltransferases↗
SEARCH · PubMed Health
Results for “Amino Acids, Neutral”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Cloning and functional characterization of a system ASC-like Na+-dependent neutral amino acid transporter.
A cDNA was isolated from mouse testis which encodes a Na+-dependent neutral amino acid transporter. The encoded protein, designated ASCT2, showed amino acid sequence similarity to the mammalian glutamate transporters (40-44% identity), Na+-dependent neutral amino acid transporter ASCT1 (57% identity; Arriza, J. L., Kavanaugh, M. P., Fairman, W. A., Wu, Y.-N., Murdoch, G. H., North, R. A., and Amara, S. G.(1993) J. Biol. Chem. 268, 15329-15332; Shafqat, S., Tamarappoo, B. K., Kilberg, M. S., Puranam, R. S., McNamara, J. O., Guadano-Ferraz, A., and Fremeau, T., Jr. (1993) J. Biol. Chem. 268, 15351-15355) and a mouse adipocyte differentiation-associated gene product AAAT (94% identity; Liao, K., and Lane, D.(1995) Biochem. Biophys. Res. Commun. 208, 1008-1015). When expressed in Xenopus laevis oocytes, ASCT2 exhibited Na+-dependent uptakes of neutral amino acids such as L-alanine, L-serine, L-threonine, L-cysteine, and L-glutamine at high affinity with Km values around 20 microM. L-Methionine, L-leucine, L-glycine, and L-valine were also transported by ASCT2 but with lower affinity. The substrate selectivity of ASCT2 was typical of amino acid transport system ASC, which prefers neutral amino acids without bulky or branched side chains. ASCT2 also transported L-glutamate at low affinity (Km = 1.6 mM). L-Glutamate transport was enhanced by lowering extracellular pH, suggesting that L-glutamate was transported as protonated form. In contrast to electrogenic transport of glutamate transporters and the other ASC isoform ASCT1, ASCT2-mediated amino acid transport was electroneutral. Na+ dependence of L-alanine uptake fits to the Michaelis-Menten equation, suggesting a single Na+ cotransported with one amino acid, which was distinct from glutamate transporters coupled to two Na+. Northern blot hybridization revealed that ASCT2 was mainly expressed in kidney, large intestine, lung, skeletal muscle, testis, and adipose tissue. Functional characterization of ASCT2 provided fruitful information on the properties of substrate binding sites and the mechanisms of transport of Na+-dependent neutral and acidic amino acid transporter family, which would facilitate the structure-function analyses based on the comparison of the primary structures of ASCT2 and the other members of the family.
Amino Acid Sequence↗
Changes in brain levels of acidic, basic, and neutral amino acids after consumption of single meals containing various proportions of protein.
Rats fasted overnight were allowed to consume single meals containing 0, 18, or 40% protein or continued to fast; after 2 h, brains and sera were taken and assayed for various amino acids. In general, serum levels of most amino acids were reduced by the 0% protein meal and elevated by the high-protein meal when compared with those associated with fasting conditions. Exceptions were those not diminished by the 0% protein meal (tryptophan, methionine, proline) and those increased (alanine) or decreased (glycine) by all of the test meals. Amino acids exhibiting the broadest normal ranges (estimated by comparing their serum levels after 40% protein with those after 0% protein) were tyrosine, leucine, valine, isoleucine, and proline; serum lysine and histidine, two basic amino acids, also varied more than threefold. Brain levels of lysine, histidine, and some of the large neutral amino acids (LNAAs) also exhibited clear relationships to the protein content of the test meal: those of valine, leucine, and isoleucine were depressed by the 0% protein but increased (compared with 0% protein) when protein was added to the meal: brain tyrosine was increased by all of the test meals in proportion to their protein contents; tryptophan, phenylalanine, and glutamate were increased after the 0% protein meal but not by protein-containing meals; brain lysine, histidine, and methionine were increased after the high-protein meal, and brain alanine was increased slightly by all of the meals.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acids↗
Diet-induced changes in plasma amino acid pattern: effects on the brain uptake of large neutral amino acids, and on brain serotonin synthesis.
Tryptophan is transported into brain by a competitive carrier system it shares with such other large neutral amino acids as tyrosine, phenylalanine, leucine, isoleucine, and valine. Physiologic variations in the plasma neutral amino acid pattern (either as a change in plasma tryptophan, or in the plasma concentration of one or more of its competitors) directly alter this competitive process, and thereby modify the uptake of tryptophan into brain. Such variations in tryptophan uptake influence brain tryptophan levels, and thus serotonin synthesis. Food intake, by influencing directly the plasma levels of large neutral amino acids, ca- therefore predictably modify brain trypotphan uptake and serotonin synthesis. The effect of food intake on the competitive uptake of tryptophan into brain, and on brain tryptophan levels, has recently been shown not to be limited to this amino acid, but also holds for other large neutral amino acids, and for certain large neutral amino acid drugs (e.g., methyldopa). Hence, following a meal, the brain concentration of any large neutral amino acid appears to depend on how the food modifies the plasma level of that amino acid relative to the plasma concentrations of its competitors. The binding of tryptophan to albumin in blood has also been suggested to influence brain tryptophan uptake. However, this notion has not been sustained by the results of nutritional studies, in which meal-induced changes in brain tryptophan levels were readily shown not to be predicted by the alterations in the size of the serum free tryptophan pool. Taken together, these data affirm the importance of competitive transport in determining brain tryptophan uptake and levels, but question whether serum albumin binding and the size of the free tryptophan pool function physiologically to modulate brain tryptophan concentrations.
Animals↗
Mammalian amino acid transport system y+ revisited: specificity and cation dependence of the interaction with neutral amino acids.
A reevaluation of the specificity of system y+, the classical transporter for cationic amino acids is presented. System y+ has been defined as a transporter for cationic amino acids that binds neutral amino acids with lower affinity in the presence of Na+. The discovery of other transporters for cationic amino has suggested that some properties, originally attributed to system y+, may relate to other transport systems. Uncertainty concerns mainly, the affinity for neutral amino acids and the cation dependence of this interaction. Neutral amino acids (13 analogues tested) were found to bind to system y+ in human erythrocytes with very low affinity. Inhibition constants (Kiy, mm) ranged between 14.2 mm and >400 mm, and the strength of interaction was similar in the presence of Na+, K+ or Li+ (145 mm). In choline medium, no interaction was detected up to 20 mm of the neutral amino acid. Guanidinium ion (5 mm, osmolarity maintained with choline) potentiated neutral amino acid binding; the effect was most important in the case of l-norvaline which aligned with guanidinium ion is equivalent to arginine. This suggests cooperative interaction at the substrate site. The specificity of system y+ was shown to be clearly distinct from that of system y+L, a cationic amino acid transporter that accepts neutral amino acids with high affinity in the presence of Na+ and which influenced the classical definition of system y+.
Amino Acid Transport Systems↗
Placental amino acid uptake. III. Transport systems for neutral amino acids.
The human placenta is known to concentrate nearly all amino acids intracellularly for transfer to the fetus. To clarify the mechanism and regulation of this process we have determined the specificity of the principal placental transport systems for neutral amino acids. With the use of competitive inhibition techniques, three transport systems of overlapping specificity have been elucidated. These correspond approximately to the "A", "L", and "ASC" systems of Christensen and associates. In the placenta the specificity of these systems is as follows: A system - alpha aminoisobutyric acid (AIB), glycine, proline, N-methylalanine, alanine, serine, threonine, and glutamine; L system - isoleucine, valine, phenylalanine, BCH, alanine, serine, threonine, and glutamine; and ASC system - alanine, serine, threonine, and glutamine. Placental AIB uptake previously has been shown to increase with preincubation of tissue in vitro. This increase has now been found to be limited to the A system. Activity of the other two systems is essentially unaffected, demonstrating that the transport pathways are separately regulated.
Amino Acids↗
Interactions between amino acid transport systems in Neurospora crassa.
Mutants of Neurospora crassa, selected as resistant to l-canavanine and l-thialysine, are partially deficient in the uptake of basic amino acids. Neutral amino acids completely inhibit uptake of basic amino acids, and this inhibition is dependent on the activity of a neutral amino acid permease. In contradistinction, mutants resistant to 4-methyl-dl-tryptophan are partially deficient in the uptake of neutral amino acids. Basic amino acids completely inhibit neutral amino acid uptake, and this inhibition is dependent on the activity of a basic amino acid permease. It is proposed that these specific transport systems compete with a general amino acid permease for some common element. The general permease is also regulated by ammonia, the amino acid pool, or both. The activity of the general permease can be eliminated phenotypically by a high concentration of glycerol or a high temperature. It is also shown that l-citrulline is transported by the neutral amino acid permease and by the general amino acid permease.
Amino Acids↗
Characterization of carrier-mediated transport systems for small neutral amino acids in human fibroblast lysosomes.
Analog inhibition studies of the uptake of proline, serine, and threonine into human fibroblast lysosomes, purified on Percoll gradients, reveal the presence of three new transport systems. These systems fail to show the Na+ requirement usual for the plasma membrane. Proline uptake into fibroblast lysosomes occurs mainly by two routes: a predominant route half-saturating at 0.01 mM, and a lower-affinity route, half-saturating at 0.07 mM. The latter so far appears specific for L-proline and its 3,4-dehydro derivative. The high affinity route has a broad scope, recognizing best, beyond these two amino acids, various unbranched neutral amino acids not over 5 carbons long. Neither system accepts to a significant extent D-proline, hydroxyproline, cationic or anionic amino acids, nor neutral ones with bulky side chains. 2-Aminoisobutyrate and its N-methyl derivative have little effect on proline uptake, in contrast to their effectiveness on its uptake by the intact fibroblast. The rate of lysosomal proline uptake maximizes at about pH 6.4, is inversely related to the osmolarity of the medium, and is unaffected by the extralysosomal presence of MgATP. The competition among alanine, serine, and threonine points to sharing of the broad-scope system for proline, although the main part of their uptake occurs by a third route that rejects amino acids in which the alpha-amino group is methylated.
Alanine↗
Neutral amino acid transport in Leishmania promastigotes.
Neutral amino acid transport was investigated in Leishmania promastigotes. Proline and alanine transport occur against their concentration gradient although there is a very rapid (40% at 30 min) conversion of proline to alanine. Uptake of these amino acids occurs by a sodium-independent route which is completely eliminated by addition of CCCP or KCN. Km values for proline and alanine are 80 microM and 63 microM with Vmax values of 6.4 and 7.2 nmol/min per mg dry weight, respectively. Countertransport of proline, alanine and phenylalanine was measured by loading the cells with a variety of neutral amino acids and proline analogs, followed by CCCP addition. The effect of aminooxyacetic acid, an inhibitor of alanine aminotransferase (EC 2.6.1.2), on proline and alanine countertransport was also examined. The results obtained are consistent with the presence of at least two systems for neutral amino acid transport in Leishmania promastigotes.
Alanine↗
Characteristics of acidic, basic and neutral amino acid transport in the perfused rat hindlimb.
1. We have employed a paired-tracer isotope dilution technique in a perfused rat hindlimb preparation to obtain information on the kinetics of transport across the sarcolemmal membranes of acidic, neutral and basic amino acids. 2. We have defined the characteristics of the saturable transport of amino acids normally regarded as paradigm substrates for the A, ASC, L, y+(basic) and the dicarboxylic amino acid transport systems. Their maximal transport capacities (Vmax, nmol min-1 (g muscle)-1 and substrate concentrations for half-maximal transport (Km, mM) of representative amino acid substrates are as follows: 2-aminoisobutyrate (AIB), Vmax = 15 +/- 7, Km = 1.26 +/- 0.6; alanine, Vmax = 332 +/- 53, Km = 3.9 +/- 0.9; serine, Vmax = 410 +/- 61, Km 3.4 +/- 0.5; leucine, Vmax = 2800 +/- 420, Km = 20 +/- 2; lysine, Vmax = 136 +/- 46, Km = 2.1 +/- 1.3; glutamate, Vmax = 86 +/- 6, Km = 1.05 +/- 0.05; proline, Vmax = 196 +/- 48, Km = 4.1 +/- 0.6. 3. Glycine uptake was faster than expected on the basis of diffusion but was not saturable and showed uptake that could be best described by a first-order rate constant of 0.07 +/- 0.003 min-1. 4. We have attempted to discriminate kinetically between possible routes of entry for an amino acid on the basis of competitive and non-competitive interaction between substrates potentially sharing common routes. On this basis, the major routes of alanine entry appear to be via the ASC and L systems with the A system playing a quantitatively minor role. Glutamate and aspartate appear to be transported exclusively by a dicarboxylate amino acid carrier. The branched-chain amino acids (BCAA) and the aromatic amino acid, phenylalanine, are almost equivalent substrates for an L-like system. 5. Insulin had no detectable effect on the uptake of paradigm substrates for ASC, L, y+, the dicarboxylic amino acid or glycine transport systems. 6. Transport of serine and lysine was Na+ dependent. Lysine transport apparently occurred with a stoichiometry of 2 Na+: 1 lysine. With the exception of alanine, whose transport was partially Na+ dependent, all other amino acids examined in the present study were transported in a Na+-independent manner.
Alanine↗
Site-directed mutagenesis of cysteine residues of large neutral amino acid transporter LAT1.
The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood-brain barrier (BBB). Owing to the high affinity (low Km) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low Km of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys160 of LAT1 and Cys110 of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl2 with a Ki of 0.56+/-0.11 microM. The inhibitory effect of HgCl2 for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl2 for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys88 and Cys439, altered amino acid transport. The Vmax was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys439 residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane.
Amino Acid Sequence↗
Structure-affinity relationships of substrates for the neutral amino acid transport system in rabbit ileum.
The apparent affinities of various amino acids for the neutral amino acid transport system in rabbit ileum were determined by measuring the inhibition of L-methionine-(14)C influx across the brush border membrane. The apparent affinity was very low for compounds lacking an alpha-amino group, compounds with the alpha-hydrogen substituted by a methyl group, D-compounds, compounds with tertiary branching in the side chain, compounds with either a positive or negative charge in the side chain, and in most cases, compounds with a hydrophilic moiety in the side chain. High apparent affinities were exhibited by compounds with unbranched carbon or carbon-sulfur side chains. Branched compounds such as valine and leucine exhibited affinities which correlate with binding of only the linear portion of the side chain. The calculated change in free energy of binding is 370 cal/mol/CH(2) group which suggests the binding region for the side chain is partially hydrophobic. The affinities of families of analogues, derivatives of cysteine, methionine, serine, alanine, valine, and phenylalanine, correlate with their calculated octanol/water partition coefficients and are also correlated with apparent structural and electronic differences between families. The data permit a preliminary description of the functional geometry of the neutral amino acid transport site. The site contains a region for binding the alpha-amino group, alpha-carboxyl group, and side chain. The regions about the alpha-amino group and alpha-hydrogen are quite sterically limited. The side chain binding region is hydrophobic in nature and appears to be shallow, binding only the linear portion of branched or ring compounds.
Amino Acids↗
Neutral amino acid transport in mouse peritoneal macrophages.
Neutral amino acid transport has been characterized in the mouse peritoneal macrophages elicited by thioglycolate broth. Serine, alanine, cysteine, and leucine were transported mainly in a Na+-independent manner and, within the concentration ranges tested, the transport of each amino acid was mediated by a single, saturable system. The transport of these amino acids was strongly inhibited by a wide variety of neutral amino acids, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid, a putative System L substrate, but not by glycine, proline, or 2-methylaminoisobutyrate. In this study the transport of serine has been characterized in detail. Inhibition of the transport of serine by 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid or by leucine was competitive. The transport of serine which exhibited trans-stimulation was slightly stimulated by lowering the pH and was little affected by N-ethylmaleimide. It was also shown that most of the neutral amino acids were concentrated only slightly (less than 2-fold) in the macrophages, unlike in the lymphocytes and granulocytes. From these results we have concluded that in the macrophages serine, cysteine, alanine, leucine, and presumably many other neutral amino acids are transported almost exclusively through a common transport system analogous to System L.
Alanine↗
Changes with aging in the levels of amino acids in rat CNS structural elements. II. Taurine and small neutral amino acids.
Taurine (Tau) and the small neutral amino acids glycine (Gly), serine (Ser), threonine (Thr), and alanine (Ala) were measured in 53 brain areas of 3- and 29-month-old male Fisher 344 rats. The ratio of highest to lowest level was 34 for Tau, 9.1 for Thr, 7.6 for Gly and Ser, and 6.5 for Ala. The heterogeneity was found in numerous areas; for example, Tau levels were more than 90 nmol/mg protein in 6 areas, and less than 20 nmol/mg protein in 10 areas. Similar heterogeneity was found with the other amino acids. The relative distribution of the small neutral amino acids showed several similarities; Tau distribution was different. With age, four amino acids decreased in 10-18 areas, and increased in only 1-3, while Thr increased in more areas than it decreased. The five amino acids of this paper, and the four of the previous paper, are among the amino acids at highest level in the brain; the sequence in their levels shows considerable regional heterogeneity.
Aging↗
Characterization of the amino acid response element within the human sodium-coupled neutral amino acid transporter 2 (SNAT2) System A transporter gene.
The neutral amino acid transport activity, System A, is enhanced by amino acid limitation of mammalian cells. Of the three gene products that encode System A activity, the one that exhibits this regulation is SNAT2 (sodium-coupled neutral amino acid transporter 2). Fibroblasts that are deficient in the amino acid response pathway exhibited little or no induction of SNAT2 mRNA. Synthesis of SNAT2 mRNA increased within 1-2 h after amino acid removal from HepG2 human hepatoma cells. The amino acid responsive SNAT2 genomic element that mediates the regulation has been localized to the first intron. Increased binding of selected members of the ATF (activating transcription factor) and C/EBP (CCAAT/enhancer-binding protein) families to the intronic enhancer was established both in vitro and in vivo. In contrast, there was no significant association of these factors with the SNAT2 promoter. Expression of exogenous individual ATF and C/EBP proteins documented that specific family members are associated with either activation or repression of SNAT2 transcription. Chromatin immunoprecipitation analysis established in vivo that amino acid deprivation led to increased RNA polymerase II recruitment to the SNAT2 promoter.
Activating Transcription Factors↗
Molecular and functional characterization of intestinal Na(+)-dependent neutral amino acid transporter B0.
We have cloned the Na(+)-dependent neutral amino acid transporter B0 (ATB0) from rabbit jejunum and from the human intestinal cell line Caco-2. Rabbit intestinal ATB0 (riATB0) cDNA codes for a protein of 541 amino acids with 10 potential transmembrane domains. When expressed in HeLa cells, riATB0 mediates the transport of several neutral amino acids, including glutamine, in a Na(+)-dependent manner. Anionic amino acids, cationic amino acids, and N-methylated amino acids are excluded by riATB0. When expressed in Xenopus laevis oocytes, riATB0 increases the transport of neutral amino acids severalfold. The induced transport activity is specific for neutral amino acids, with no noticeable interaction with anionic, cationic, and N-methylated amino acids. However, riATB0 does interact with anionic amino acids at acidic pH. In oocytes expressing riATB0, the neutral amino acid threonine evokes inward currents at a holding potential of -50 mV. The amino acid-evoked current is sensitive to membrane potential. The inward current increases as the membrane potential is hyperpolarized, but the current reverses at about -30 to -40 mV. Threonine evokes outward currents if the membrane potential is depolarized beyond this value. We have also cloned the ATB0 from the human intestinal cell line Caco-2. The Caco-2 ATB0 cDNA also codes for a protein of 541 amino acids that is essentially identical to the ATB0 expressed in the human choriocarcinoma cell line JAR. Reverse transcription-polymerase chain reaction (RT-PCR) and restriction analysis of the RT-PCR products indicate that the human intestine and the human kidney proximal tubular cell line HKPT express an ATB0 identical to the ATB0 expressed in Caco-2 cells.
Amino Acid Sequence↗
Increased plasma concentrations of the 5-HT precursor amino acid tryptophan and other large neutral amino acids in violent criminals.
BACKGROUND: Aggression has been suggested to be related to abnormal brain 5-HT activity. This amine is synthesized in brain from tryptophan, which is transported through the blood-brain barrier in competition with other amino acids. The relationship between tryptophan and its endogenous amino acid competitors in plasma might thus influence the availability of tryptophan in the brain and consequently brain 5-HT activity. METHODS: Plasma amino acids were determined in 89 offenders who had committed various violent and non-violent crimes and in 14 healthy controls. RESULTS: Both tryptophan and its competitors were higher in offenders who had committed violent crimes compared both with non-violent offenders and with controls. No difference was, however, seen in the relationship between tryptophan and its competitors. CONCLUSIONS: The results support the contention that violent behaviour is related to biochemical deviations but could not explain a possible decreased brain 5-HT activity.
Adult↗
The role of blood-brain barrier transport of tryptophan and other neutral amino acids in the regulation of substrate-limited pathways of brain amino acid metabolism.
Many pathways of essential neutral amino acid metabolism in the CNS are influenced by precursor availability. Since the delivery of circulating amino acids to brain cells is primarily controlled by the rate of amino acid transport through the blood-brain barrier (BBB), pathways of brain amino acid metaboliransport system. The Km of BBB transport is in the 0.1--0.6 mM range, which approximates the physiologic plasma levels and forms the basis of the unusual sensitivity of the brain to competition effects on neutral amino acid transport. Unlike the brain, the Km of amino acid transport into other organs is in the 1--10 mM range or greater, which frees these tissues from competition effects in the physiologic range of plasma amino acids. Tryptophan circulates 80--90% bound to albumin; however, the capacity/affinity ratio of the BBB neutral amino acid transport system exceeds the capacity/affinity ratio of albumin binding of tryptophan, which enables the carrier to strip tryptophan off albumin as it traverses the brain capillary. The activity of the BBB neutral amino acid transport system is probably not modulated by insulin, but is influenced by changes in thyroid hormone status; the transport system is also induced in states of hepatic encephalopathy and this induction process is the primary cause of the increased brain tryptophan and serotonin levels in cirrhosis.
Animals↗