The transport of L-alanine by the hamster kidney cell line BHK-21-C13.

The uptake of L-alanine into BHK21-C13 cells in culture has been studied. This amino acid appears to be transported essentially via a relatively low affinity, high capacity, sodium ion dependent transport system. Inhibition studies using other amino acids or their analogues provided information about the specificity of this system. This alanine transport system was shown to exhibit a broad substrate specificity and appeared to be capable of transporting most naturally occurring neutral alpha-amino acids. Kinetic studies of the inhibition of L-alanine uptake also indicated the presence of a second neutral amino acid transport system capable of transporting this amino acid. However, it is unlikely that this second uptake system contributes greatly to L-alanine uptake. Inhibition of the uptake of L-leucine indicated that this transport system has a similar specificity to the "L"-system initially described for Ehrlich ascites carcinoma cells.     

Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides.

A proton motive force-driven di-tripeptide carrier protein (DtpT) and an ATP-dependent oligopeptide transport system (Opp) have been described for Lactococcus lactis MG1363. Using genetically well-defined mutants in which dtpT and/or opp were inactivated, we have now established the presence of a third peptide transport system (DtpP) in L. lactis. The specificity of DtpP partially overlaps that of DtpT. DtpP transports preferentially di- and tripeptides that are composed of hydrophobic (branched-chain amino acid) residues, whereas DtpT has a higher specificity for more-hydrophilic and charged peptides. The toxic dipeptide L-phenylalanyl-beta-chloro-L-alanine has been used to select for a di-tripeptide transport-negative mutant with the delta dtpT strain as a genetic background. This mutant is unable to transport di- and tripeptides but still shows uptake of amino acids and oligopeptides. The DtpP system is induced in the presence of di- and tripeptides containing branched-chain amino acids. The use of ionophores and metabolic inhibitors suggests that, similar to Opp, DtpP-mediated peptide transport is driven by ATP or a related energy-rich phosphorylated intermediate.     

The acidic amino acid transport system of the baby hamster kidney cell line BHK21-C13

The uptake of L-glutamate into BHK21-C13 cells in culture has been studied. This amino acid appears to be transported via a relatively high affinity, low capacity, Na+-dependent transport system capable of the rapid accumulation of substrate amino acids. Kinetic studies of the inhibition of L-glutamate uptake has provided information as to the substrate and the molecular configuration required for transport via the glutamate transport system. This system exhibited marked substrate specificity and was only capable of transporting L-glutamate and aspartate and certain closely related acidic amino acid analogues.     

A general amino-acid permease is inducible in Chlorella vulgaris

Glucose or non-metabolizable glucose analogues induce two systems of amino-acid transport in Chlorella vulgaris: an arginine-lysine system and a proline system. An additional third system of amino-acid transport is induced when glucose and an inorganic nitrogen source are present during glucose induction. The transport rates in glucose-NH ₄ ⁺ -treated cells are 10 to 80 times higher than in untreated cells. The transport system shows a rather broad specificity and catalyses the transport of at least ten neutral and acidic amino acids. Three of these amino acids (l-alanine, l-serine and glycine) are transported by the proline system as well. The system is specific for l-amino acids and has a pH optimum between 5 and 6. Transport by this system seems to be active, since amino acids are accumulated inside the cells.     

Characterization of tryptophan transport in human placental brush-border membrane vesicles.

The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity.     

Detection and characterization of carrier-mediated cationic amino acid transport in lysosomes of normal and cystinotic human fibroblasts. Role in therapeutic cystine removal?

The discovery of a trans-stimulation property associated with lysine exodus from lysosomes of human fibroblasts has enabled us to characterize a system mediating the transport of cationic amino acids across the lysosomal membrane of human fibroblasts. The cationic amino acids arginine, lysine, ornithine, diaminobutyrate, histidine, 2-aminoethylcysteine, and the mixed disulfide of cysteine and cysteamine all caused trans-stimulation of the exodus of radiolabeled lysine from the lysosomal fraction of human fibroblasts at pH 6.5. In contrast, neutral and acidic amino acids did not affect the rate of lysine exodus. trans-Stimulation of lysine exodus was observed over the pH range from 5.5 to 7.6, was specific for the L-isomer of the cationic amino acid, and was intolerant to methylation of the alpha-amino group of the amino acid. The lysosomotropic amine, chloroquine, greatly retarded lysine exodus, whereas the presence of sodium ion was without effect. The specificity and lack of Na+ dependence of this lysosomal transport system is similar to that of System y+ present on the plasma membrane of human fibroblasts. In addition, we find cystine exodus from the lysosomal fraction of cystinotic human fibroblasts to be greatly retarded as compared to that of normal human fibroblasts with half-times of exodus similar to those reported for the lysosomes of cystinotic and normal human leukocytes (Gahl, W. A., Tietze, F., Bashan, N., Steinherz, R., and Schulman, J. D. (1982) J. Biol. Chem. 257, 9570-9575). In contrast, normal and cystinotic human fibroblasts did not show any differences with regard to lysine efflux or its trans-stimulation by cationic amino acids. An important mechanism by which cysteamine treatment of cystinosis allows cystine escape from lysosomes may be the ability of the mixed disulfide of cysteine and cysteamine formed by sulfhydryl-disulfide exchange to migrate by this newly discovered system mediating cationic amino acid transport.     

Characterization of L-threonine and L-glutamine transport in murine P388 leukemia cells in vitro. Presence of an N-like amino acid transport system

The transport of L-threonine and L-glutamine into murine P388 leukemia cells has been characterized. Threonine appears to be a specific substrate for a Na+-dependent amino acid transport system similar to system ASC of the HTC hepatoma cell. Threonine transport is uninhibited by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid and alpha-(methylamino)isobutyric acid, shows a pattern of transport similar to that seen in HTC hepatoma cells over the pH range of 5.5-7.5, and is inhibited by L-serine and L-cysteine. Approximately two-thirds of glutamine transport into P388 cells also appears to enter P388 cells via this ASC-analogous system. However, based upon (a) inhibition studies with threonine (where the K1 of threonine inhibition of glutamine transport was 7-fold the Km of threonine transport), (b) inhibition analysis of glutamine transport with various amino acids and amino acid analogues, and (c) different patterns of transport between threonine and glutamine over the pH range of 5.5-7.5, approximately one-third of glutamine transport can be attributed to a second Na+-dependent amino acid transport system. This system appears to be similar to the system N of rat hepatocytes. Glutamine and threonine do not appear to enter P388 cells via systems A or L to any significant degree. P388 cells do not appear to exhibit 'adaptive regulation' of amino acid transport. Differences in 'adaptive regulation' could therefore not be utilized for comparing threonine and glutamine transport.     

Enhanced transport of natural amino acids after activation of pig lymphocytes.

Na+-dependent uptake of the amino acids L-proline and L-methionine was greatly accelerated when pig lymphocytes were activated with phytohaemagglutinin or other mitogens. The increased influx was apparent after incubation with phytohaemagglutinin for 1 h, and reached a maximum after 24 h. The lymphocytes appear to possess at least three different transport systems for neutral amino acids with properties similar to, but not identical with, those described for other cells. The activity of a system resembling the A system of other cells was increased most dramatically after activation, its activity in unstimulated lymphocytes being extremely low or absent. A second Na+-dependent system, which has properties similar to those of the ASC system in other cells, but with a broader specificity for amino acids, was more active in unstimulated lymphocytes, and uptake by this system was also accelerated after incubation with phytohaemagglutinin. The activity of a third system, very similar to the L system in other cells, was increased to a much smaller extent after lymphocyte activation.     

Cloning and functional characterization of a Na(+)-independent, broad-specific neutral amino acid transporter from mammalian intestine

We have isolated a cDNA from a rabbit intestinal cDNA library which, when co-expressed with the heavy chain of the human 4F2 antigen (4F2hc) in mammalian cells, induces system L-like amino acid transport activity. This protein, called LAT2, consists of 535 amino acids and is distinct from LAT1 which also interacts with 4F2hc to induce system L-like amino acid transport activity. LAT2 does not interact with rBAT, a protein with a significant structural similarity to 4F2hc. The 4F2hc/LAT2-mediated transport process differs from the 4F2hc/LAT1-mediated transport in substrate specificity, substrate affinity, tissue distribution, interaction with D-amino acids, and pH-dependence. The 4F2hc/LAT2-associated transport process has a broad specificity towards neutral amino acids with K(t) values in the range of 100-1000 microM, does not interact with D-amino acids to any significant extent, and is stimulated by acidic pH. In contrast, the 4F2hc/LAT1-associated transport process has a narrower specificity towards neutral amino acids, but with comparatively higher affinity (K(t) values in the range of 10-20 microM), interacts with some D-amino acids with high affinity, and is not influenced by pH. LAT2 is expressed primarily in the small intestine and kidney, whereas LAT1 exhibits a much broader tissue distribution.     

The transamination of L-cystathionine, L-cystine and related compounds by a bovine kidney transaminase

An enzyme which actively transaminates L-cystathionine, L-cystine, L-lanthionine and S-aminoethyl-L-cysteine has been purified from bovine kidney. The transaminase appears to be pure up to 90% and probably consists of two subunits of similar molecular mass of about 47 kDa. The enzymatic products arising from the transamination of L-cystathionine and related compounds spontaneously cyclize into ketiminic structures, which are the immediate precursors of unusual imino acids recovered in biological materials. The specificity towards other amino acid and oxo acid acceptors is similar to the specificity exhibited by rat kidney glutamine transaminase. This suggests that the sulfur amino acid transaminations that have been described could be performed by the bovine kidney glutamine transaminase.     

Isolation and function of the amino acid transporter PAT1 (slc36a1) from rabbit and discrimination between transport via PAT1 and system IMINO in renal brush-border membrane vesicles

Reabsorption of amino acids is an important function of the renal proximal tubule. pH-dependent amino acid transport has been measured previously using rabbit renal brush-border membrane vesicles (BBMV). The purpose of this investigation was to determine whether this pH-dependent uptake represents H(+)/amino acid cotransport via a PAT1-like transport system. The rabbit PAT1 cDNA was isolated (2296bp including both 5' and 3' untranslated regions and poly(A) tail) and the open reading frame codes for a protein of 475 amino acids (92% identity to human PAT1). Rabbit PAT1 mRNA was found in all tissues investigated including kidney. When expressed heterologously in a mammalian cell line, rabbit PAT1 mediates pH-dependent, Na(+)-independent uptake of proline, glycine, l-alanine and alpha-(methylamino)isobutyric acid. Proline uptake was maximal at pH 5.0 (K(m) 2.2+/-0.7 mM). A transport system with identical characteristics (ion dependency, substrate specificity) was detected in rabbit renal BBMV where an overshoot was observed in the absence of Na+ but in the presence of an inwardly directed H+ gradient. In the presence of Na+ and under conditions in which PAT1 transport function was suppressed, a second proline uptake system was detected that exhibited functional characteristics similar to those of the IMINO system. The functional characteristics of rabbit PAT1 in either mammalian cells or renal BBMV suggest that PAT1 is the low-affinity transporter of proline, glycine and hydroxyproline believed to be defective in patients with iminoglycinuria.     

Substrate specificity and transport mode of the proton-dependent amino acid transporter mPAT2.

The PAT2 transporter has been shown to act as an electrogenic proton/amino acid symporter. The PAT2 cDNA has been cloned from various human, mouse and rat tissues and belongs to a group of four genes (pat1 to pat4) with PAT3 and PAT4 still resembling orphan transporters. The first immunolocalization studies demonstrated that the PAT2 protein is found in the murine central nervous system in neuronal cells with a proposed role in the intra and/or intercellular amino acid transport. Here we provide a detailed analysis of the transport mode and substrate specificity of the murine PAT2 transporter after expression in Xenopus laevis oocytes, by electrophysiological techniques and flux studies. The structural requirements to the PAT2 substrates - when considering both low and high affinity type substrates - are similar to those reported for the PAT1 protein with the essential features of a free carboxy group and a small side chain. For high affinity binding, however, PAT2 requires the amino group to be located in an alpha-position, tolerates only one methyl function attached to the amino group and is highly selective for the L-enantiomers. Electrophysiological analysis revealed pronounced effects of membrane potential on proton binding affinity, but substrate affinities and maximal transport currents only modestly respond to changes in membrane voltage. Whereas substrate affinity is dependent on extracellular pH, proton binding affinity to PAT2 is substrate-independent, favouring a sequential binding of proton followed by substrate. Maximal transport currents are substrate-dependent which suggests that the translocation of the loaded carrier to the internal side is the rate-limiting step.     

A family of yeast proteins mediating bidirectional vacuolar amino acid transport

Seven genes in Saccharomyces cerevisiae are predicted to code for membrane-spanning proteins (designated AVT1-7) that are related to the neuronal gamma-aminobutyric acid-glycine vesicular transporters. We have now demonstrated that four of these proteins mediate amino acid transport in vacuoles. One protein, AVT1, is required for the vacuolar uptake of large neutral amino acids including tyrosine, glutamine, asparagine, isoleucine, and leucine. Three proteins, AVT3, AVT4, and AVT6, are involved in amino acid efflux from the vacuole and, as such, are the first to be shown directly to transport compounds from the lumen of an acidic intracellular organelle. This function is consistent with the role of the vacuole in protein degradation, whereby accumulated amino acids are exported to the cytosol. Protein AVT6 is responsible for the efflux of aspartate and glutamate, an activity that would account for their exclusion from vacuoles in vivo. Transport by AVT1 and AVT6 requires ATP for function and is abolished in the presence of nigericin, indicating that the same pH gradient can drive amino acid transport in opposing directions. Efflux of tyrosine and other large neutral amino acids by the two closely related proteins, AVT3 and AVT4, is similar in terms of substrate specificity to transport system h described in mammalian lysosomes and melanosomes. These findings suggest that yeast AVT transporter function has been conserved to control amino acid flux in vacuolar-like organelles.     

Characterization of neutral amino acid transport in a marine pseudomonad.

The transport of neutral amino acids in marine pseudomonad B-16 (ATCC 19855) has been investigated. From patterns of competitive inhibition, mutant analysis, and kinetic data, two active transport systems with overlapping substrate specificities were distinguished and characterized. One system (DAG) served glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid (AIB) and, to a lesser extent, L-alanine and possibly other related neutral D- and L-amino acids. The other system (LIV) showed high stereospecificity for neutral amino acids with the L configuration and served primarily to transport L-leucine, L-isoleucine, L-valine, and L-alanine. This system exhibited low affinity for alpha-aminoisobutyric acid. Neither system was able to recognize structural analogues with modified alpha-amino or alpha-carboxyl groups. The kinetic parameters for L-alanine transport by the DAG and LIV systems were determined with appropriate mutants defective in either system. For L-alanine, Kt values of 4.6 X 10(-5) and 1.9 X 10(-4) M and Vmax values of 6.9 and 20.8 nmol/min per mg of cell dry weight were obtained for transport via the DAG and LIV systems respectively. alpha-Aminoisobutyric acid transport heterogeneity was also resolved with the mutants, and Kt values of 2.8 X 10(-5) and 1.4 X 10(-3) M AIB were obtained for transport via the DAG and LIV systems, respectively. Both systems required Na+ for activity (0.3 M Na+ optimal) and in this regard are distinguished from systems of similar substrate specificity reported in nonmarine bacteria.     

Neutral amino acid transport in isolated rat pancreatic islets

The neutral amino acid transport systems of freshly isolated rat pancreatic islets have been studied by first examining the transport of L-alanine and the nonmetabolizable analogue 2-(methylamino)isobutyric acid (MeAIB). By comparing the uptake of MeAIB and L-alanine for their pH dependency profile, choline and Li+ substitution for Na+, tolerance to N-methylation, and competition with other amino acids, the existence in pancreatic islets of both A and ASC amino acid transport systems was established. The systems responsible for the inward transport of five natural amino acids was studied using competition analysis and Na+ dependency of uptake. These studies defined three neutral amino acid transport systems: A and ASC (Na+-dependent) and L (Na+-independent). L-Proline entered rat islet cells mainly by system A; L-leucine by the Na+-independent system L. The uptake of L-alanine, L-serine, and L-glutamine was shared by systems ASC and L, the participation of system A being negligible for these three amino acids. An especially broad substrate specificity for systems L and ASC is therefore suggested for the rat pancreatic islet cells. The regulation of amino acid transport was also investigated in two conditions differing as to glucose concentration and/or availability, i.e. islets from fasted rats and islets maintained in tissue culture at high or low glucose concentrations. Neither alanine nor MeAIB transport was altered by fasting of the islet-donor rats. On the other hand, pancreatic islets maintained for 2 days in tissue culture at high (16.7 mM) glucose transported MeAIB at twice the rate of islets maintained at low (2.8 mM) glucose. Amino acid starvation of pancreatic islets during 11 h of tissue culture resulted in a 2-fold increase in MeAIB transport.     

Substrate recognition by the mammalian proton-dependent amino acid transporter PAT1

The PAT family of proton-dependent amino acid transporters has recently been identified at the molecular level. This paper describes the structural requirements in substrates for their interaction with the cloned murine intestinal proton/amino acid cotransporter (PAT1). By using the Xenopus laevis oocytes as an expression system and by combining the two-electron voltage clamp technique with radiotracer flux studies, it was demonstrated that the aliphatic side chain of L-alpha-amino acids substrates can consist maximally of only one CH2-unit for high affinity interaction with PAT1. With respect to the maximal separation between the amino and carboxyl groups, only two CH2-units, as in gamma-aminobutyric acid (GABA), are tolerated. PAT1 displays no or even a reversed stereoselectivity, tolerating serine and cystein only in the form of D-enantiomers. A methyl-substitution of the carboxyl group (e.g. O-methyl-glycine) markedly diminishes substrate affinity and transport rates, whereas methyl-substitutions at the amino group (e.g. sarcosine or betaine) have only minor effects on substrate interaction with the transporter binding site. Furthermore, it has been shown (by kinetic analyses of radiolabelled betaine influx and inhibition studies) that the endogenous PAT system of human Caco-2 cells has very similar transport characteristics to mouse PAT1. In summary, one has defined the structural requirements and limitations thet determine the substrate specificity of PAT1. A critical recognition criterion of PAT1 is the backbone charge separation distance and the side chain size, whereas substitutions on the amino group are well tolerated.     

Specificity and regulation of peptide transport on Neurospora crassa.

The tripeptide, glycyl-d,l-leucyl-l-tyrosine was chemically synthesized in radioactive form and used to directly study the specificity, regulation, and properties of an oligopeptide transport system in Neurospora. Transport activity is sensitive to azide but does not result in the accumulation of the intact peptide; rather, the radioactive label is accumulated as free tyrosine. Inhibition studies suggest that the transport system probably has a relatively wide range of specificity and is responsible for uptake of short oligopeptides of quite distinct sequences. However, free amino acids and dipeptides are not transported significantly, if at all, by the oligopeptide transport system. A free amino group appears to be a requirement for peptide transport. A mutant strain that is unable to use various peptides for growth is further described and shown to be reduced greater than 90% in transport of the tripeptide.     

Characteristics of the Amino Acid Transport System in the Mucosal Border of Rabbit Ileum

The specificity of the neutral amino acid transport system in the brush border was examined by studying the ability of amino acid analogues to inhibit the unidirectional influx of phenylalanine from mucosal solution into the cells. Effects were evaluated in terms of the affinity of various substrates for the amino acid site in the transport system. The affinity of amino acids for the site was proportional to the number of carbon atoms in the side chain. Electron-withdrawing substituents in the ring of phenylalanine increased affinity and electron-releasing groups decreased affinity. Removal of the α-amino group from phenylalanine decreased affinity by a factor of approximately 50 and removal of the carboxyl group decreased affinity 12-fold. Effects on affinity of variations in the side chain of the amino acid can be comparable in magnitude to that of the carboxyl group. The effect of sodium ion on the transport system appears to be similar for all compounds tested.     

Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of Escherichia coli

The nucleotide sequence of the genes encoding the high affinity, branched-chain amino acid transport systems LIV-I and LS has been determined. Seven genes are present on a 7568-base pair DNA fragment, six of which participate directly in branched-chain amino acid transport. Two periplasmic amino acid-binding proteins are encoded by the livJ (LIV-BP) and livK (LS-BP) genes. These two proteins confer specificity on the LIV-I and LS transport systems. livK is the first gene in a polycistronic message that includes four genes encoding membrane components, livHMGF. The protein products of the livHMGF genes are shared by the two systems. An analysis of the livH and livM DNA sequences suggests that they encode hydrophobic proteins capable of spanning the membrane several times. The LivG and LivF proteins are less hydrophobic, but are also tightly associated with the membrane. Both LivG and LivF contain the consensus sequence for adenine nucleotide binding observed in many other transport proteins. A deletion strain that does not express any of the liv genes was constructed. This strain was used to show that each of the membrane component genes is required for high affinity leucine transport, including two genes, livM and livF, for which no previous genetic evidence had been obtained.     

Developmental regulation of amino acid transport in Neurospora crassa

Conidia of Neurospora crassa exhibit an ability to transport various amino acids against a concentration gradient. The conidial transport system has previously been characterized in terms of kinetics, competitions, and genetic control. This study describes the development of a new and highly active transport capability which is elaborated during the early stages of development but prior to evident germination. It has been named “postconidial” transport activity and represents as much as 20-fold greater initial rates as compared to those observed with conidia. Development of the postconidial transport activity requires protein synthesis and can be partially repressed when the substrate amino acid is present during the developmental preincubation period. A mutant has been utilized which exhibits normal conidial but fails to develop normal postconidial transport activity for any amino acid examined. Although temperature optimum and pH dependence are similar in conidial and postconidial systems, there is evidence that the new activity is not a simple amplification of an existing capability. This is reflected as a change in competition patterns between particular amino acids as development proceeds.