Role of proton dissociation in the transport of acidic amino acids by the Ehrlich ascites tumor cells

The pH profile for the uptake of l-glutamic acid by the Ehrlich ascites tumor cell arises largely as a sum of the decline with falling pH of a slow, Na⁺-dependent uptake by System A, and an increasing uptake by Na⁺-independent System L. The latter maximizes at about pH 4.5, following approximately the titration curve of the distal carboxyl group. This shift in route of uptake was verified by (a) a declining Na⁺-dependent component. (b) an almost corresponding decline in the 2-(methylamino)-isobutyric acid-inhibitable component, (c) a rising component inhibited by 2-aminonorbornane-2-carboxylic acid. Other amino acids recognized as principally reactive with Systems A or L yielded corresponding inhibitory effects with some conspicuous exceptions: 2-Aminoisobutyric acid and even glycine become better substrates of System L as the pH is lowered; hence their inhibitory action on glutamic acid uptake is not lost. The above results were characterized by generally consistent relations among the half-saturation concentrations of the interacting amino acids with respect to: their own uptake, their inhibition of the uptake, one by another, and their trans stimulation of exodus, one by another.A small Na⁺-dependent component of uptake retained by l-glutamic acid but not by d-glutamic acid at pH 4.5 is inhibitable by methionine but by neither 2-(methylamino)-isobutyric acid nor the norbornane amino acid. We provisionally identified this component with System ASC, which transports l-glutamine throughout the pH range studied. No transport activity specific to the anionic amino acids was detected, and the unequivocally anionic cysteic acid showed neither significant mediated uptake nor inhibition of the uptake of glutamic acid or of the norbornane amino acid.     

Characterization of neutral and cationic amino acid transport in Xenopus oocytes

Amino acid transport was characterized in stage 6 Xenopus laevis oocytes. Most amino acids were taken up by the oocytes by way of both Na+-dependent and saturable Na+-independent processes. Na+-dependent transport of 2-aminoisobutyric acid (AIB) was insensitive to cis- or trans-inhibition by the System A-defining substrate 2-(methylamino)-isobutyric acid (MeAIB), although threonine, leucine, and histidine were found to be effective inhibitors, eliminating greater than 80% of Na+-dependent AIB uptake. Lack of inhibition by arginine eliminates possible mediation by System Bo,+ and suggests uptake by System ASC. The Na+-dependent transport of characteristic System ASC substrates such as alanine, serine, cysteine, and threonine was also insensitive to excess MeAIB. Evidence to support the presence of System Bo,+ was obtained through inhibition analysis of Na+-dependent arginine transport as well arginine inhibition of Na+-dependent threonine uptake. The Na+-independent transport of leucine was subject to trans-stimulation and was inhibited by the presence of excess phenylalanine, histidine, and, to a lesser extent, 2-amino-(2,2,1)-bicycloheptane-2-carboxylic acid (BCH). These observations are consistent with mediation by System L. The characteristics of Na+-independent uptake of threonine are not consistent with assignment to System L, and appear to be reflective of Systems asc and bo,+. In its charged state, histidine appears to be transported by a carrier similar in its specificity to System y+, but is taken up by System L when present as a zwitterion.     

Heterogeneity of histidine transport in the Ehrlich cell.

We have reexamined the heterogeneity shown by histidine in its uptake by the Ehrlich ascites tumor cell, in the face of a contradiction of our earlier interpretation. We again find the fraction of histidine uptake at neutral pH inhibitable by the model substrate for System A, 2-(methylamino)-isobutyric acid, to be fully dependent on the presence of Na+ or Li+. The small Na+ -independent component not attributable to System L can be identified with System Ly+ through its inhibitability by homoarginine. This component increases as the pH is lowered with an apparent pK' a of 6.1. The simultaneous decrease in the uptake by the neutral systems could be identified, for System L, with the same titration of histidine to its cationic form, but for System A the sharp decrease is identified with the protonation of a structure on the membrane rather than one on the substrate. The action of H+ in the latter case proved approximately non-competitive with Na+ when tested with ordinary substrates.     

Stimulation of Na+ -dependent amino acid uptake by activation of the Ca2+ -dependent K+ channel in the Ehrlich ascites tumor cell

The activation of Ca2+ -dependent K+ channel by propranolol or by ascorbate-phenazine methosulphate stimulates Na+ -dependent transport of alpha-aminoisobutyric acid. This stimulation arises from a membrane hyperpolarization due to the specific increase of membrane K+ conductance. The same treatment does not modify the Na+ -independent uptake of the norbornane amino acid.     

Comparison of system N in fetal hepatocytes and in related cell lines

In contrast to the changes seen in membrane transport systems for other neutral, anionic, and cationic amino acids, System N for glutamine, histidine, and asparagine in the rat hepatocytes shows nearly constant properties at the fetal, differentiated, and cultured hepatoma stages. These properties were tested by measuring the Na+-dependent transport of glutamine. This approximate constancy applies not only to the transport selectivity of the system among neutral amino acids, but also to its tolerance of Li+ as a substitute for Na+, its characteristic sensitivity to pH lowering, its relative sensitivity to N-ethylmaleimide, its stimulation by amino acid deprivation, and its failure to respond to insulin or glucagon. The properties of histidine as a substrate for System N were also examined. Inhibition studies with different cell types suggest that the Na+-dependent glutamine and histidine uptake is more restricted to System N in the hepatoma line H35 (H4-11-EC,3) and in the fetal hepatocyte than in hepatoma line HTC and the Ehrlich cells. The Na+-independent component of glutamine and histidine uptake was greater in the hepatoma cells in continuous culture than in fetal and adult hepatocytes in primary culture. Trans-stimulation of glutamine and histidine influx into H35 cells occurs predominantly by the Na+-independent route.     

Structural determinants in substrate recognition by proton-amino acid symports in plasma membrane vesicles isolated from sugar beet leaves.

Amino acids are actively transported across the plasma membrane of plant cells by proton-coupled symports. Previously, we identified four amino acid symports in isolated plasma membrane vesicles, including two porters for the neutral amino acids. Here we investigated the effect of amino acid analogues on neutral amino acid transport to identify structural features that are important in molecular recognition by Neutral System I (isoleucine) and Neutral System II (alanine and leucine). D-Isomers of alanine and isoleucine were not effective transport antagonists of the L-isomers. These data are characteristic of stereospecificity and suggest that the positional relationship between the alpha-amino and carboxyl groups is an important parameter in substrate recognition. This conclusion was supported by the observation that beta-alanine and analogues with methylation at the alpha-carbon, at the carboxyl group, or at the alpha-amino group were not effective transport inhibitors. Specific binding reactions were also implicated in these experiments because substitution of the alpha-amino group with a space filling methyl or hydroxyl group eliminated transport inhibition. In contrast, analogues with various substitutions at the distal end of the amino acid were potent antagonists. Moreover, the relative activity of several analogues was influenced by the location of sidechain branches and Neutral Systems I and II were resolved based on differential sensitivity to branching at the beta-carbon. The kinetics of azaserine and p-nitrophenylalanine inhibition of leucine transport were competitive. We conclude that the binding site for the carboxyl end of the amino acid is a well-defined space that is characterized by compact, asymmetric positional relationships and specific ligand interactions. Although the molecular interactions associated with the distal portion of the amino acid were less restrictive, this component of the enzyme-substrate complex is also important in substrate recognition because the neutral amino acid symports are able to discriminate between specific neutral amino acids and exclude the acidic and basic amino acids.     

Discrimination of Na+-independent transport systems L, T, and asc in erythrocytes. Na+ independence of the latter a consequence of cell maturation?

On the basis of inhibition analysis two bicyclic amino acid analogs appear to enter human red blood cells by much the same Na+-independent mediation, whereas striking differences are apparent in the routes for tryptophan and leucine, confirming a role for System T, but also suggesting the participation of a third system of low affinity somewhat selective for weakly basic amino acids. System T of the human cell is specifically inhibited by 4-azidophenylalanine, and is highly sensitive, relative to System L, to N-ethylmaleimide inhibition. Uptake by System T approaches its steady state much more slowly than does System L, and its participation in trans-stimulation is questionable, whereas that of System L is as usual strong. A different added transport system became apparent in the slow approach of the Na+-independent mediation of uptake of 3- and 4-carbon dipolar amino acids by the nucleated pigeon red cell to its steady state. In that cell System T makes at most a minor contribution. The patterns of trans-stimulation of fluxes among selected pairs of amino acids in the pigeon cell correspond to a usual participation in transmembrane exchange by System L, and also by the new transport system. An important but not the sole source of the heterogeneity in the pigeon cell is the participation of the system conspicuously involved in the transport of alanine, serine, and threonine, among other amino acids. This route of transport of these amino acids is made conspicuous by their small transport by other Na+-independent agencies, notably System L. Reactivity with this system is enhanced by a side change hydroxyl or sulfhydryl group. Uptake by this route as tested by threonine showed little inhibition by cysteinesulfinate under conditions inhibitory to System asc; also a sensitivity to lowering of pH unlike that seen with System asc. The new Na+-dependent transport system appears to be a species variant of quite similar Na+-independent systems discovered by Young et al. (Young, J. D., Ellory, J. C., and Tucker, E. M. (1975) Nature (Lond.) 254, 156-157; Fincham, D. A., Mason, D. K., and Young, J. D. (1982) Biochem. Soc. Trans. 11, 776-777) in sheep and horse erythrocytes on the basis of their absence in phenotypes. These authors have emphasized several similarities in these two cases to Na+-dependent System asc, and they propose that Na+ dependence has specifically been lost on maturation of the red cells without major changes in amino acid selectivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characteristics of glutamic acid transport by rabbit intestinal brush-border membrane vesicles. Effects of Na+-, K+- and H+-gradients

In the presence of a Na+-gradient (out greater than in), L-glutamic acid and L-and D-aspartic acids were equally well concentrated inside the vesicles, while no transport above simple diffusion levels was seen by replacement of Na+ by K+. Equilibrium uptake values were found inversely proportional to the medium osmolarity, thus demonstrating uptake into an osmotically sensitive intravesicular space. The extrapolation of these lines to infinite medium osmolarity (zero space) showed only a small binding component in acidic amino-acid transport. When the same experiment was performed at saturating substrate concentrations, linear relationships extrapolating through the origin but showing smaller slope values were recorded, thus indicating that the binding component could be more important than suspected above. However, binding to the membrane was neglected in our studies as it was absent from initial rate measurements. Na+-dependent uphill transport of L-glutamic acid was stimulated by K+ present on the intravesicular side only but maximal stimulation was recorded under conditions of an outward K+-gradient (in greater than out). Quantitative and qualitative differences in the K+ effect were noted between pH 6.0 and 8.0. Initial uptake rates showed pH dependency in Na+-(out greater than in) + K+-(in greater than out) gradient conditions only with a physiological pH optimum between 7.0 and 7.5. It was also found that a pH-gradient (acidic outside) could stimulate both the Na+-gradient and the Na+ + K+-gradient-dependent transport of L-glutamic acid. However, pH- or K+-gradient alone were ineffective in stimulating uptake above simple diffusion level. Finally, it was found that increased rates of efflux were always observed with an acidic pH outside, whatever the conditions inside the vesicles. From these results, we propose a channel-type mechanism of L-glutamic acid transport in which Na+ and K+ effects are modulated by the surrounding pH. The model proposes a carrier with high or low affinity for Na+ in the protonated or unprotonated forms, respectively. We also propose that K+ binding occurs only to the unprotonated carrier and allows its fast recycling as compared to the free form of the carrier. Such a model would be maximally active and effective in the intestine in the in vivo physiological situations.     

Heterogeneity of transport systems for L-glutamine in mouse mammary gland

The characteristics of the transport systems of L-glutamine in lactating mouse mammary gland have been studied. L-glutamine uptake was mediated by three Na+-dependent and one Na+-independent systems. The 2-(methylamino)isobutyric acid-sensitive component of Na+-dependent uptake exhibited the usual characteristics of system A. The other two Na+-dependent systems, which we have named BCI(-)-dependent and BCl(-)-independent, are the new systems identified. These are broad specificity systems and were discriminated on the basis of inhibition analysis, Cl- dependency and the effect of preloading mammary tissue with amino acids. While L-aspargine inhibited the uptake of L-glutamine via both these broad specificity systems, L-homoserine inhibited the uptake of L-glutamine via only BCl(-)-dependent system. The uptake of L-glutamine via the BCl(-)-independent system was upregulated by preloading mammary tissue with L-serine, while BCl(-)-dependent system was unaffected. The Na+-independent uptake of L-glutamine was inhibited by 2-aminobicyclo-(2,2,1)heptane carboxylic acid and other neutral amino acids, and identified as the system L.     

Acidic amino acid transport characteristics of a newly developed conditionally immortalized rat type 2 astrocyte cell line (TR-AST).

To characterize acidic amino acid transport in type 2 astrocytes, we established conditionally immortalized rat astrocyte cell lines (TR-AST) from newly developed transgenic rats harboring temperature-sensitive SV40 large T-antigen gene. TR-AST exhibited positive immunostaining for anti-GFAP antibody and A2B5 antibody, characteristics associated with type 2 astrocytes, and expressed glutamine synthetase. Acidic amino acid transporters, GLT-1 and system xc-, which consists of xCT and 4F2hc, were expressed in all TR-ASTs by RT-PCR. On the other hand, GLAST expression was found in TR-AST3 and 5. The characteristics of [3H]L-glutamic acid (L-Glu) uptake by TR-AST5 include an Na+-dependent and Na+-independent manner, concentration-dependence, and inhibition by L-aspartic acid (L-Asp) and D-aspartic acid (D-Asp). The corresponding Michaelis-Menten constants for the Na+-dependent and Na+-independent process were 36.3 microM and 155 microM, respectively. [3H]L-Asp and [3H]D-Asp uptake by TR-AST5 had an Na+-dependent and Na+-independent manner. This study demonstrated that GLT-1, system xc-, and GLAST were expressed in TR-AST, which has the characteristics of type 2 astrocytes and is able to transport acidic amino acids.     

Consequences of aspartase deficiency in Yersinia pestis.

Growing cells of Yersinia pseudotuberculosis, but not those of closely related Yersinia pestis, rapidly destroyed exogenous L-aspartic and L-glutamic acids, thus prompting a comparative study of dicarboxylic amino acid catabolism. Rates of amino acid metabolism by resting cells of both species were determined at pH 5.5, 7.0, and 8.5. Regardless of pH, Y. pseudotuberculosis destroyed L-glutamic acid, L-glutamine, L-aspartic acid, and L-asparagine at rates greater than those observed for Y. pestis. Although rates of proline degardation were similar, its metabolism by Y. pestis at pH 8.5 resulted in excretion of glutamic and aspartic acids. Similarly, Y. pestis excreted aspartic acid when incubated with L-glutamic acid (pH 8.5) or L-asparagine (pH 5.5, 7.0, and 8.5). Aspartase activity was not detected in extracts of 10 strains of Y. pestis but was present in all 11 isolates of Y. pseudotuberculosis. The latter contained significantly more glutaminase, asparaginase, and L-glutamate-oxalacetate transminase activity than did extracts of Y. pestis; specific activities of L-glutamate dehydrogenase and alpha-ketoglutarate dehydrogenase were similar. The observed differences in dicarboxylic amino acid metabolism are traceable to asparatase deficiency in Y. pestis and may account for the slow doubling time of this organism relative to Y. pseudotuberculosis.     

Characterization of amino acid transport during erythroid cell differentiation

We have studied the changes in amino acid transport in fetal erythroid cells isolated from rat fetal liver at different gestation days. Our results show that System A transport as measured by the Na+-dependent uptake of 2-(methylamino)isobutyric acid (MeAIB) was conspicuous at day 13 but virtually disappeared between days 16 and 18. In contrast, the activity of System ASC measured by the Na+-dependent uptake of MeAIB-insensitive threonine uptake increased after day 14 and was optimal between days 16 and 18. This transport system regressed in activity with further maturation, but remained conspicuously saturable in the matured red blood cell. Interestingly, the newly discovered Na+-independent System asc (Vadgama, J. V., and Christensen, H.N. (1985) J. Biol. Chem. 260, 2912-2921), selective for the uptake of test substrates threonine, serine, and alanine, was present in these erythroid cells. Its activity increased during gestation days 16-18. System L transport was present simultaneously with the Na+-independent System asc. As we had previously demonstrated for the pigeon red blood cell, these two transport systems are kinetically independent as confirmed with inhibition studies and the special selectivity of System L to trans stimulation. Tryptophan uptake could be attributed predominantly to System L, as also observed for the nucleated pigeon red blood cells and certain other cells. Arginine showed its familiar Na+-independent mode of uptake as a cation throughout the interval of study. An exceptional Na+-dependent component of arginine uptake emerged after day 14, peaked at day 18, and then disappeared on further maturation of the erythroid cell.     

Effect of L-Aspartic Acid and L-Glutamic Acid on Production of L-Proline

To elucidate the effect of aspartic acid on growth of Kurthia catenaforma during the proline fermentation, this organism was compared with other bacteria with respect to the rate of consumption of aspartic acid, and to the activities of enzymes concerned in the metabolism of aspartic acid. Although no marked difference in enzyme activities was observed, the aspartic acid consumption rate of K. catenaforma was markedly higher than that of other organisms. The consumption of glutamic acid by K. catenaforma was not detected at 24 hr of culture. The difference between the consumption of aspartic acid and glutamic acid in this strain might result from a difference in permeability to the amino acids. We considered that L-glutamic acid might substitute for L-aspartic acid if the uptake of glutamic acid could be increased. A number of detergents were screened for their effect on consumption of glutamic acid. Cetyltrimethylammonium bromide, sodium laurylphosphate, and polyoxyethylene sorbitan monolaurate were found to increase the transport rate of glutamic acid, but not of aspartic acid. A method of producing L-proline from glutamic acid was established with the aid of detergents.     

The transport of alanine, serine, and cysteine in cultured human fibroblasts

The transport of L-alanine, L-serine, and L-cysteine has been studied in skin-derived diploid human fibroblasts in culture. Competition analysis, mathematical discrimination by nonlinear regression, and conditions varying the relative contribution of the various mediations have been used to characterize the systems engaged in the inward transport of these amino acids. All the adopted criteria yielded results showing that L-alanine, L-serine, and L-cysteine enter the cell by two Na+-dependent systems, System A and System ASC, and by a Na+-independent route, whose major component has been identified as System L. The apparent affinity of L-alanine, L-serine, and L-cysteine for the putative carrier was higher for System ASC than for System A. The transport Vmax for System A increased in response to cell starvation; after 12 h, its values were similar or higher than those exhibited by System ASC. At amino acid concentrations approaching those present in human plasma, System ASC appeared to be the primary mediation for the inward transport of L-alanine, L-serine, and L-cysteine in human fibroblasts. The contribution of System A was negligible in nonstarved cells and became appreciable under conditions of cell starvation. The Na+-independent System L made no substantial contribution to the uptake of L-alanine and L-serine and accounted for approximately one-fourth of the total uptake of L-cysteine.     

Kinetics and Specificity of Amino Acid Uptake by the Duckweed Spirodela polyrhiza (L.) Schleiden

Borstlap, A. G, Meenks, J. L. D., van Eck, W. F. and Bicker,J. T. E. 1986. Kinetics and specificity of amino acid uptakeby the duckweed Spirodela polyrhiza (L.) Schleiden.—J.exp. Bot. 37: 1020–1035. Uptake of ¹⁴C-labelled amino acids by intact, axenically grownplants of Spirodela polyrhiza (L.) Schleiden was investigated.Experiments in which uptake was measured from the decrease inthe amino acid concentration in the medium, indicated that saturableuptake conforms to the sum of two Michaelis-Menten terms, possiblycorresponding with a high-affinity and a low-affinity system.Further experiments with L-leucine, L-glutamic acid, and L-lysine,in which uptake was measured by assaying the amount of ¹⁴ inthe plants, showed the presence of a non-saturable componentin addition to the dual saturable uptake. Uptake of L-glutamic acid precipitously declined between pH4?0 and 6? and that of L-leucine between pH 4?0 and 8? whereasL-lysine uptake was optimal at pH 6?0. No evidence was foundthat the apparent high-affinity and low-affinity systems respondeddifferently to changes in external pH or to the addition ofCCCP. The non-saturable uptake component was not affected bychanges in external pH or by adding CCCP, and might have beendue to free space uptake. Mutual inhibition of uptake was found between acidic and neutralamino acids (L-leucine, L-methionine, L-glutamic acid) and betweenbasic amino acids (L-lysine, L-ornithine). The basic amino acidshad no effect on the uptake of L-leucine, L-methionine and L-glutamicacid, although the uptake of basic amino acids was inhibitedby glutaminc acid and several neutral amino acids. It is suggested that the duckweed has a high-affinity transportsystem for neutral and acidic amino acids, and a distinct high-affinitysystem for basic amino acids. It is argued that the first systemtransports zwitterionic amino acids (z-system), and that thesecond system transports cationic amino acids(y⁺-system). Thespecificity of the low-affinity system is less certain, butthere is some evidence that it is similar to that of their high-affinitycounterparts. Key words: Kinetics, membrane transport, pH-dependency, transport systems, uptake isotherms     

pH-dependent heterogeneity of acidic amino acid transport in rabbit jejunal brush border membrane vesicles.

Initial rates of Na(+)-dependent L-glutamic and D-aspartic acid uptake were determined at various substrate concentrations using a fast sampling, rapid filtration apparatus, and the resulting data were analyzed by nonlinear computer fitting to various transport models. At pH 6.0, L-glutamic acid transport was best accounted for by the presence of both high (Km = 61 microM) and low (Km = 7.0 mM) affinity pathways, whereas D-aspartic acid transport was restricted to a single high affinity route (Km = 80 microM). Excess D-aspartic acid and L-phenylalanine served to isolate L-glutamic acid flux through the remaining low and high affinity systems, respectively. Inhibition studies of other amino acids and analogs allowed us to identify the high affinity pathway as the X-AG system and the low affinity one as the intestinal NBB system. The pH dependences of the high and low affinity pathways of L-glutamic acid transport also allowed us to establish some relationship between the NBB and the more classical ASC system. Finally, these studies also revealed a heterotropic activation of the intestinal X-AG transport system by all neutral amino acids but glycine through an apparent activation of Vmax.     

Neutral amino acid transport in embryonal carcinoma cells

Neutral amino acid transport was characterized in the pluripotent embryonal carcinoma (EC) cell line, OC15. Ten of the thirteen amino acids tested are transported by all three of the major neutral amino acid transport systems--A, L, and ASC--although one system may make a barely measurable contribution in some cases. The characterization of N-methyl-aminoisobutyric acid (meAIB) transport points to this model amino acid as a definitive substrate for System A transport by OC15 cells. Thus, high concentrations of meAIB can be used selectively to block System A transport, and the transport characteristics of meAIB represent system A transport. Kinetic analysis of System A, with a Km = 0.79mM and Vmax = 14.4 nmol/mg protein/5 min, suggests a single-component transport system, which is sensitive to pH changes. While proline transport in most mammalian cells is largely accomplished through System A, it is about equally divided between Systems A and ASC in OC15 cells, and System A does not contribute at all to proline transport by F9 cells, an EC cell line with limited developmental potential. Kinetic analysis of System L transport, represented by Na+-independent leucine transport, reveals a high-affinity, single-component system. This transport system is relatively insensitive to pH changes and has a Km = 0.0031 mM and Vmax = 0.213 nmol/mg protein/min. The putative System L substrate, 2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH), inhibits Systems A and ASC as well as System L in OC15 cells. Therefore, BCH cannot be used as a definitive substrate for System L in OC15 cells. Phenylalanine is primarily transported by Na+-dependent Systems A and ASC (83% Na+-dependent; 73% System ASC) in OC15 cells, while it is transported primarily by the Na+-independent System L in most other cell types, including early cleavage stage mouse embryos and F9 cells. We have also found this unusually strong Na+-dependency of phenylalanine transport in mouse uterine blastocysts (82% Na+-dependent). There is no evidence for System N transport by OC15 cells, since histidine is transported primarily by a Na+-independent, BCH-inhibitable mechanism.     

Amino acid transport by the filamentous fungus Arthrobotrys conoides

Uptake of l-valine by germinated spores of Arthrobotrys conoides has all the characteristics of a system of transport that requires an expenditure of energy by the cells. It is dependent on temperature and has an energy of activation of 16,000 cal/mole. Uptake is optimal at pH 5 to 6. l-Valine accumulated against a concentration gradient and is not lost from the cells by leakage or exchange. The process requires energy supplied by the metabolic reactions that are inhibited by catalytic amounts of 2,4-dinitrophenol and azide. The kinetics of the system are consistent with a mechanism of transport that depends on a limited number of sites on the cell surface, and the Michaelis constant for the system is 1.5 x 10(-5) to 7.5 x 10(-5)m. Modification of the amino or carboxyl group abolishes l-valine uptake. The process is competitively inhibited by d-valine, glycine, and other neutral amino acids (K(i) = 1.5 x 10(-5) to 4.0 x 10(-5)m), indicating a lack of stereospecificity, and also indicating that aliphatic side chain is not required for binding with the carrier. The transport system has less affinity for acidic amino acids (glutamic and aspartic acids) than neutral amino acids, and a greater affinity for basic amino acids (histidine, lysine, and arginine). The range of affinity is in the order of 100, as measured in terms of K(i) values for various compounds. The data presented provide suggestive evidence that the uptake by A. conoides of all amino acids except proline is mediated by a single carrier system that possesses an overall negative charge.     

Important differences in cationic amino acid transport by lysosomal system c and system y+ of the human fibroblast

Superficial similarities led us to extend our designation for the transport of the plasma membrane for cationic amino acids, y+, to the lysosomal system also serving for such amino acids. Further study on the purified lysosomes of human skin fibroblasts leads us now to redesignate the lysosomal system as c (for cationic), rather than y+, to emphasize important contrasts. Lysosomal uptake of arginine at pH 7.0 was linear during the first 2 min, but attained a steady state in 6 min. This arginine uptake was Na+-independent and was tripled in rate when the lysosomes had first been loaded with the cationic amino acid analog, S-2-aminoethyl-L-cysteine. Uptake was slowed to one-third when 2 mM MgATP was added to the incubation mixture. The following differences in cationic amino acid influx between lysosomal System c and the plasma membrane System y+ became apparent: 1) arginine influx is increased 10-fold by raising the external pH from 5.0 to 7.0. This effect favors net entry of cationic amino acids under the H+ gradient prevailing in vivo. In contrast, arginine uptake across the plasma membrane is insensitive to pH changes in this range. 2) The Km of arginine uptake by lysosomal System c, 0.32 mM, is eight times that for System y+ arginine uptake by the fibroblast. 3) Certain neutral amino acids in the presence of Na+ are accepted as surrogate substrates by System y+, but not by lysosomal system c. 4) Cationic amino acids in which the alpha-amino group is monomethylated or the distal amino group is quaternary, also D-arginine, are recognized by lysosomal System c, whereas System y+ has little affinity for these analogs. This broader substrate specificity of lysosomal system c led us to discover that thiocholine serves to deplete accumulated cystine from cystinotic fibroblasts as effectively as does the therapeutic agent, cysteamine. The quaternary nitrogen of thiocholine renders the mixed disulfide formed when it reacts with cystine unsatisfactory as a substrate for System y+.     

Homocysteine uptake by human umbilical vein endothelial cells in culture

The characteristics of the uptake of L-homocysteine by cultures of human umbilical vein endothelial cells have been examined. Uptake occurred by Na(+)-dependent and Na(+)-independent systems, but was essentially independent of the pH of the uptake medium. The Na(+)-independent system corresponded to system L, being totally inhibited by the presence of beta-2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) a system L analogue. It was concluded on the basis of starvation experiments coupled with failure to detect any inhibition in the presence of 2-methylaminoisobutyric acid (MeAIB), a system A analogue, that the Na(+)-dependent uptake was wholly accounted for by system ASC. The kinetic properties of systems L and ASC were determined by omitting Na+ from the uptake medium and incorporating BCH in the medium, respectively. It has been concluded on the basis of the inhibitory effects of a number of amino acids that uptake of homocysteine occurs by those systems which transport cysteine.