Amino Acid Transport Processes Research Articles

Effects of the Fenton reagent on transport in yeast.

In the facultatively anaerobic yeast Saccharomyces cerevisiae the uptake rate and the accumulation ratio of 2-aminoisobutyric acid was decreased by some 30% by Fenton's reagent (FR), a powerful source of OH. radicals. Likewise, the uptake of glutamic acid, leucine and arginine was diminished. The mediated diffusion of 6-deoxy-D-glucose was not affected. The H+ symport of maltose and trehalose was inhibited by some 40% both in the initial rate and in the accumulation ratio. FR had a dramatic inhibitory effect when present during preincubation with 50 mmol/L glucose. In the obligately aerobic Lodderomyces elongisporus the uptake of all amino acids tested was decreased by 15-30%, that of 6-deoxy-D-glucose by about 10%. The initial rates of uptake of maltose and trehalose were depressed by FR by 40% and the acceleration of uptake observed after 8 min of incubation, was abolished by FR completely. Acidification rate of the external medium by S. cerevisiae in the presence of glucose or galactose was enhanced three-fold, that after subsequently added K+ was substantially decreased. FR appears to have a dual effect on sugar and amino acid transport processes in yeast: (1) it blocks carrier protein synthesis; (2) it inhibits the source of energy for transport. It does not appreciably affect the carrier proteins themselves.

Cloning and Expression of a b0,+-like Amino Acid Transporter Functioning as a Heterodimer with 4F2hc Instead of rBAT: A NEW CANDIDATE GENE FOR CYSTINURIA

We have cloned a transporter protein from rabbit small intestine, which, when coexpressed with the 4F2 heavy chain (4F2hc) in mammalian cells, induces a b(0,+)-like amino acid transport activity. This protein (4F2-lc6 for the sixth member of the 4F2 light chain family) consists of 487 amino acids and has 12 putative transmembrane domains. At the level of amino acid sequence, 4F2-lc6 shows significant homology (44% identity) to the other five known members of the 4F2 light chain family, namely LAT1 (4F2-lc1), y(+)LAT1 (4F2-lc2), y(+)LAT2 (4F2-lc3), xCT (4F2-lc4), and LAT2 (4F2-lc5). The 4F2hc/4F2-lc6 complex-mediated transport process is Na(+)-independent and exhibits high affinity for neutral and cationic amino acids and cystine. These characteristics are similar to those of the b(0,+)-like amino acid transport activity previously shown to be associated with rBAT (protein related to b(0,+) amino acid transport system). However, the newly cloned 4F2-lc6 does not interact with rBAT. This is the first report of the existence of a b(0,+)-like amino acid transport process that is independent of rBAT. 4F2-lc6 is expressed predominantly in the small intestine and kidney. Based on the characteristics of the transport process mediated by the 4F2hc/4F2-lc6 complex and the expression pattern of 4F2-lc6 in mammalian tissues, we suggest that 4F2-lc6 is a new candidate gene for cystinuria.

Amino Acid Transport through Cation Exchange Membranes: Effects of pH on Interfacial Transport

We present a series of experiments especially designed to show the effects of pH on the interfacial transport of glycine through a cation exchange membrane. The experimental results are explained qualitatively by using a theoretical model which incorporates the equilibrium dissociation equations for the fraction of amino acid forms at different pH values, a rate equation for the membrane interfacial transport based on the interfacial chemical reaction, and the Nernst–Planck flux equations for the ion transport through the bulk of the membrane. The results appear to indicate that the rate-limiting step in the amino acid transport process could be the rate at which glycine exits the membrane, and they contribute to a better understanding of amino acid permeation across charged membranes.

Effect of low-protein diet-induced intrauterine growth retardation on rat placental amino acid transport.

Given the central role of the placenta in nutrient transport to the fetus, one might propose that maternal nutrition would have a regulatory effect on this nutrient delivery. We have examined the effect of a low-protein adequate-calorie diet on specific amino acid transport processes by the rat placenta. Maternal weight, fetal weight, and placental weight were all significantly reduced in dams fed a low-protein (5% casein), isocaloric diet when compared with dams pair-fed a control (20% casein) diet. Even though maternal serum amino acid levels were maintained in the low-protein animals, fetomaternal serum amino acid ratios were significantly reduced, suggesting a reduction in nutrient transfer to the fetus. Apical and basal membrane vesicles were isolated from the placental trophoblast and were used to examine the amino acid transport capacity of both maternal-facing and fetal-facing membranes, respectively. Na+-dependent neutral amino acid transport mediated by system A was decreased in both membrane preparations, while transport mediated by system ASC was unaffected. The Na+-dependent anionic amino acid uptake by system X(-)AG (EAAC1) was reduced on the basal membrane, while the Na+-independent component was similar between the low-protein and control diet-fed dams. Cationic amino acid uptake was also reduced on both membrane surfaces. A decreased steady-state mRNA content for EAAC1 and CAT1 (system y+) suggests that reduced synthesis of the transporter proteins is responsible for the decrease in transport activity. Taken together, these data support the hypothesis that maternal protein malnutrition affects nutrient delivery to the fetus by downregulation of specific amino acid transport proteins.

Transport of cationic and zwitterionic amino acids in preimplantation rat conceptuses

The ability of preimplantation rat conceptuses to take up several amino acids was examined under a variety of conditions, and the characteristics of uptake were compared to those determined previously for mouse conceptuses. Mediated leucine transport in two-cell rat conceptuses is Na +-independent and inhibited almost completely by 2-amino- endobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), so it resembles system L which predominates in two-cell mouse conceptuses. System L becomes less conspicuous than homoarginine-sensitive, Na +-independent leucine transport (provisionally designated system b O,+) by the time rat conceptuses develop into blastocysts, as is also the case for mouse conceptuses. In contrast to leucine transport, system b O,+ appears to be the most conspicuous transporter of cationic amino acids throughout preimplantation development of both species. A Na +-independent cation-preferring amino acid transport process also appears to be present in rat as well as in mouse conceptuses. Moreover, rat conceptuses resemble mouse conceptuses because Na +-dependent transport system Gly activity virtually disappears from them by the time they form blastocysts. Unlike mouse conceptuses, however, Na +-dependent system B O,+ activity appears to be present throughout preimplantation development of rat conceptuses, whereas it has not been detected until at least the two-cell stage in the mouse. Although system B O,+ becomes more conspicuous in mouse than in rat conceptuses by the time they form blastocysts, system B O,+ activity appears to increase when blastocysts of both species are removed from the uterus just prior to implantation. The latter observation is consistent with the possibility that system B O,+ activity is controlled, in part, by the uterus near the time of implantation, although further studies are needed to verify this possibility. Similarities as well as differences in the amino acid transport processes present in conceptuses of rats and mice may eventually be understood best in relation to the environments in which they develop in vitro and in situ.

Functional changes in cation-preferring amino acid transport during development of preimplantation mouse conceptuses

In a previous study, a Na +-independent, cation-preferring amino acid transport system was detected in preimplantation mouse blastocysts. The system resisted Na +-dependent inhibition by homoserine and so resembled the lysosomal system c more than it resembled the plasmalemmal system y +. We now report the presence of a cation-preferring system in unfertilized and fertilized eggs and cleavage-state conceptuses which also resists Na +-dependent inhibition by homoserine. The systems in 1-cell conceptuses and blastocysts are, however, insensitive to changes in pH in the interval of 6.0 to 8.0 and, thus, different from the pH-sensitive system c. Moreover, the relative strengths of the interactions of a variety of basic amino acids with the systems in conceptuses do not correspond well with the relative strengths of their interactions with either system c or system y +. Similarly, the system in 1-cell conceptuses can be distinguished from the system in blastocysts because l-arginine interacts about equally well with each of these systems, whereas the system in 1-cell conceptuses is inhibited more strongly than the system in blastocysts by most other basic amino acids. In addition, inhibition of the system in 1-cell conceptuses by some basic amino acids is Na +-stimulated, whereas Na + does not affect inhibition of the system in blastocysts. Finally, l-tryptophan inhibits the system in blastocysts better than l-histidine or d-arginine do, but the reverse is true for the system in 1-cell conceptuses. Therefore, the relative activities of at least two forms of a novel, cation preferring amino acid transport process change during development of blastocysts from fertilized eggs. For convenience, the forms of the cation-preferring transport processes that seem to predominate at the 1-cell and blastocysts stages are provisionally designated systems b 1 + and b 1 +, respectively, although these two systems need not represent entirely different gene products.

Inhibition of transport system b 0,+ in blastocysts by inorganic and organic cations yields insight into the structure of its amino acid receptor site

The most conspicuous, Na +-independent amino acid transport process in preimplantation mouse blastocysts was provisionally designated system b 0,+ because it accepts some cationic and zwitterionic amino acids about equally well as substrates. Although system b 0,+ is not Na +-stimulated, it has not been determined if it is inhibited by Na +, or if its activity is affected by most other ions. Therefore, we measured uptake of amino acids by blastocysts in isotonic solutions of different ionic and nonionic osmolites. Na +-independent l-leucine uptake was unaffected by the ion concentration, but l-lysine transport was several-fold faster in isotonic solutions of non-electrolytes than in similar solutions of inorganic and organic salts or zwitterions. The K m value for ‘Na +-independent’ l-lysine transport was about 10-fold higher in isotonic salt solutions than in solutions of nonionic osmolites, whereas the K m value for l-leucine transport was about the same in either type of solution. Therefore, inorganic and organic cations and the cationic portions of zwitterions appear to compete with cationic but not zwitterionic amino acids for system b 0,+ receptor sites. The cation, harmaline, was a particularly strong competitive inhibitor of ‘Na +-independent’ l-lysine uptake by system b 0,+, even in isotonic salt solutions, whereas it inhibited l-leucine uptake noncompetitively. Moreover, harmaline appeared to compete with inorganic cations for the lysine receptor sites of system b 0,+. Harmaline also has been found by other investigators to competitively inhibit l-lysine uptake by the Na +-independent system asc 1 in horse erythrocytes, whereas it noncompetitively inhibits alanine uptake by the same system. Similarly, harmaline noncompetitively inhibits l-alanine uptake by the Na +-dependent system ASC in human erythrocytes, but it appears to compete for binding with l-alanine's cosubstrate, Na +. In addition, others have found that the positively-charged side chains of cationic amino acids seem to take the place of Na + needed near side chains in order for zwitterionic amino acids to be transported by systems ASC and y +. We conclude that system b 0,+ may be similar to systems asc 1, ASC and y +, and that each of these systems may be a variant of the same ancestral transport process. We speculate that since it appears to accept a broader scope of substrates and to interact with a wider variety of cations than do systems asc 1, ASC or y +, system b 0,+ may more closely resemble the proposed ancestral transport process than any of the other contemporary systems.

Effects of chronic ethanol treatment on amino acid uptake and enzyme activities in the lactating rat mammary gland

The effects of chronic ethanol consumption on mammary gland amino acid uptake at the 15th day of lactation in the rat have been studied. Ethanol treatment decreased the arterial levels of Ala, Asp, Gly, Pro, Lys and Met, and increased those of Gln and α-amino-butyrate. Chronic ethanol treatment produced a decrease in the arteriovenous differences of Asp, Thr, Arg, Met and Phe, and increased those of Ala, Gln, Gly, Pro and Tyr. The combination of the calculated values of relative extraction and the arteriovenous differences indicate that these alterations in amino acid uptake are related to changes in the transport process for Ala, Asp, Thr, Pro, Arg, Asn, Gly, Tyr, and Phe, and that the alterations in the arteriovenous differences of Gln, Lys and Met are due to the affected arterial levels of these amino acids. Measurements of enzymatic activities in the mammary gland show that these alterations in the amino acid transport process cannot be ascribed to changes in the α-glutamyl cycle.

Heterogeneity of sodium-dependent excitatory amino acid uptake mechanisms in rat brain.

The pharmacologic and kinetic characteristics of sodium-dependent uptake of [3H]L-glutamate, [3H]D-aspartate, and [3H]L-aspartate into crude synaptosomal preparations of rat corpus striatum and cerebellum have been examined in vitro. In cerebellum the apparent Kts and Vmax for the three excitatory amino acids were identical whereas in striatal synaptosomes, the Vmax for [3H]L-glutamate was 30% greater (P less than or equal to .001) than for [3H]D-aspartate and 50% greater (P less than or equal to .001) than for [3H]L-aspartate. L-Amino adipic acid inhibited the uptake of the three amino acids in both regions of brain was 15- to 20-fold more potent in cerebellum than in striatum. In contrast, dihydrokainic acid inhibited transport processes in the corpus striatum but was without activity in cerebellar preparations. The neurotoxin kainic acid blocked only a portion (60%) of [3H]L-glutamate and [3H]D-aspartate uptake in cerebellum while completely inhibiting amino acid transport in corpus striatum. Three days post kainic acid lesion, [3H]D-aspartate uptake was attenuated more than [3H]L-glutamate uptake in the corpus striatum; destruction of corticostriatal afferents reduced [3H]L-glutamate to a greater extent than [3H]D-aspartate. Various lesions of the cerebellum affected excitatory amino acid transport processes to a similar extent. These results suggest that excitatory amino acid transport systems are pharmacologically distinct in different brain regions and may be heterogeneous within a single region.

Amino acid supply to individual cerebral structures in awake and anesthetized rats.

The movement of phenylalanine from plasma into various cerebral structures was examined in detail in normal alert rats as well as in rats anesthetized with nitrous oxide, halothane, and sodium pentobarbital. Radioactive phenylalanine was infused intravenously in such a way as to rapidly establish and maintain a trace concentration in arterial blood. The rat was decapitated after 1.5 min, the brain removed, frozen, and thin sections cut for quantitative autoradiography. The plasma concentrations of phenylalanine as well as other neutral amino acids that compete with phenylalanine for entry into brain were measured. Phenylalanine influx varied considerably between structures, ranging from 5 nmol.min-1.g-1 in the globus pallidus to 12 nmol.min-1.g-1 in the inferior colliculus. Anesthetics reduced phenylalanine influx, but this effect was primarily due to alterations in the circulating neutral amino acid pattern. The anesthetics had little, if any, direct effect on the neutral amino acid transport process itself.

Amino acid transport

A Review of Amino Acid Transport Processes in Animal Cells and Tissues. By J. Lerner. Pp. 234. (University of Maine at Orono Press: Orono, Maine, 1978.)

Characteristics of proline transport into R3230AC mammary tumor cells

Cells separated by enzyme treatment of the R3230AC mammary carcinoma were used to characterize the entry of proline. These cells showed minimal changes in cell viability and intracellular volume and were found to be suitable for transport studies, since the v i of proline was maintained for at least 4 h when cells were stored at 37 or 4°C, or when transport was measured in the presence or absence of Na +. Proline was actively transported by these tumor cells, reaching a distribution ratio ([proline] intracellular/[proline] extracellular) of 20 after 2 h. Proline entry consisted of two processes, one saturable (carrier mediated) and the other, non-saturable. The carrier-mediated entry, K m = 0.83 mM and V = 151 · 10 −5 μmol/min per 5 · 10 6 was Na +-dependent, sensitive to pH and metabolic inhibitors, and completely inhibited by α-(methylamino)-isobutyric acid ( K i = 0.34 mM). Proline entry in the absence of Na + was 20% that in the presence of Na + and was found to be due to a non-saturable process, since (a) υ i of proline uptake in the absence of Na + increased linearly with increasing proline concentration and (b) was not suppressed by either 20 mM α-(methylamino)-isobutyric acid, 50 mM glycine + 20 mM phenylalanine, or 50 mM serine + 20 mM phenylalanine when proline uptake was measured in the presence or absence of Na +. Therefore, under the conditions studied, we conclude that proline transport appears to be restricted to the A (alanine-preferring) system. Furthermore, these cells should provide a suitable model to study the effect of hormonal manipulations on the amino acid transport process.

Characterization of active dipeptide transport by germinating barley embryos: Effects of pH and metabolic inhibitors

A sensitive new fluorescent method is outlined for the study of peptide and amino acid transport processes. Studies on dipeptide transport in germinating barley (Hordeum vulgare.) embryos have provided evidence for accumulation of substrate against a concentration gradient, saturation kinetics, a pH optimum at pH 3.8, and inhibition by a variety of metabolic inhibitors. Acetate also inhibits transport, possibly by disrupting H(+) gradients across the plasma membrane.

Active amino acid and sugar transport by a unicellular blue-green alga

1. 1. The non-metabolizable amino acid, α-aminoisobutyric-3- 14C acid, like the non-metabolizable sugar, 3-O-methyl- 14C-D-glucose, was actively transported by dark-treated “starved” Synechococcus cedrorum cells in the presence of light. 2. 2. Various unlabelled amino acids inhibited α-aminoisobutryic-3- 14C acid transport and various unlabelled sugars inhibited 3-O-methyl- 14C-D-glucose transport. 3. 3. No reciprocal inhibition, however, was observed between the active amino acid and sugar transport processes.