Acid In Mammalian Cells Research Articles

A role for thyroid hormone transporters in transcriptional regulation by thyroid hormone receptors.

Thyroid hormones (THs) must be taken up by target cells to act at the genomic level through binding to nuclear thyroid hormone receptors (TRs). Extensive study has been made of mechanisms by which TH-bound TRs regulate transcription, yet little is known about the critical upstream step, i.e. how THs enter the cell. Growing evidence suggests that saturable transport mechanisms mediate the greater part of TH movement across the plasma membrane and have important roles in the regulation of TH bioavailability. For example, System L is a multifunctional transport system serving as a plasma membrane transporter of THs and amino acids in mammalian cells. We have used two complementary systems, the Xenopus oocyte (which has negligible basal System L activity) and the mammalian BeWo cell line (which has System L activity for TH transport), to investigate the role of this representative TH transporter in nuclear action of THs. We demonstrate that overexpression of System L in Xenopus oocytes increases both cytoplasmic and nuclear delivery of THs from external medium and also enhances transcriptional activation by TRs. Conversely, blocking endogenous System L activity in BeWo cells with specific inhibitors reduces both TH uptake and TR function. These results indicate that plasma membrane TH transporters such as System L may have important roles in gene regulation by TRs.

Intracellular Trafficking and Membrane Targeting Mechanisms of the Human Reduced Folate Carrier in Mammalian Epithelial Cells

The major pathway for cellular uptake of the water-soluble vitamin folic acid in mammalian cells is via a plasma membrane protein known as the reduced folate carrier (RFC). The molecular determinants that dictate plasma membrane expression of RFC as well as the cellular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to image the targeting and trafficking dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the polypeptide and not within the cytoplasmic NH(2)- or COOH-terminal domains of the protein. Further, the integrity of the hRFC backbone is critical for export of the polypeptide from the endoplasmic reticulum to the cell surface. This trafficking is critically dependent on intact microtubules because microtubule disruption inhibits motility of hRFC-containing vesicles as well as final expression of hRFC in the plasma membrane. For the first time, these data define the mechanisms that control the intracellular trafficking and cell surface localization of hRFC within mammalian epithelia.

22ND AMINO ACID IDENTIFIED

NATURE USES ONLY A HANDful of amino acid building blocks to generate immense complexity from a relatively simple genetic code. There are just 20 genetically encoded amino acids in mammalian cells; a few organisms use 21. Now, researchers at Ohio State University have discovered a 22nd [ Science , 296,1459 and 1462 (2002)]. The team, led by microbiology professor Joseph A. Krzycki and chemistry and biochemistry professor Michael K. Chan, calls the amino acid L-pyrrolysine. The novel residue is an amide-linked 4-substituted pyrroline-5-carboxylate lysine derivative. Chan and graduate student Bing Hao identified pyrrolysine in the 1.55-A crystal structure of monomethylamine methyltransferase (MtmB) from the microbe Methanosarcina barkeri , found at the bottom of freshwater lakes. An enzyme in the pathway that makes methane from methylamine, MtmB has a barrel protein fold with pyrrolysine tucked into the bottom. But finding a novel amino acid in a protein structure doesn't necessarily mean the residue is ...

Versatile Biosynthetic Engineering of Sialic Acid in Living Cells Using Synthetic Sialic Acid Analogues

Sialic acids are critical components of many glycoconjugates involved in biologically important ligand-receptor interactions. Quantitative and structural variations of sialic acid residues can profoundly affect specific cell-cell, pathogen-cell, or drug-cell interactions, but manipulation of sialic acids in mammalian cells has been technically limited. We describe the finding of a previously unrecognized and efficient uptake and incorporation of sialic acid analogues in mammalian cells. We added 16 synthetic sialic acid analogues carrying distinct C-1, C-5, or C-9 substitutions individually to cell cultures of which 10 were readily taken up and incorporated. Uptake of C-5- and C-9-substituted sialic acids resulted in the structural modification of up to 95% of sialic acids on the cell surface. Functionally, binding of murine sialic acid-binding immunoglobulin-like lectin-2 (Siglec-2, CD22) to cells increased after N-glycolylneuraminic acid treatment, whereas 9-iodo-N-acetylneuraminic acid abolished binding. Furthermore, susceptibility to infection by the B-lymphotropic papovavirus via a sialylated receptor was markedly enhanced following pretreatment of host cells with selected sialic acid analogues including 9-iodo-N-acetylneuraminic acid. This novel experimental strategy allows for an efficient biosynthetic engineering of surface sialylation in living cells. It is versatile, extending the repertoire of modification sites at least to C-9 and enables detailed structure-function studies of sialic acid-dependent ligand-receptor interactions in their native context.

Elevated Expression of Free Deaminoneuraminic Acid in Mammalian Cells Cultured in Mannose-Rich Media

Deaminoneuraminic acid (KDN, 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) is a member of the family of sialic acids in which an acylamino group at the C-5 position of N-acylneuraminic acid (Neu5Acyl) is replaced by a hydroxyl group. It has recently been shown that KDN is synthesized de novo from its precursor, mannose (Man), in trout testis (Angata, T., Nakata, D., Matsuda, T., Kitajima, K., and Troy, F. A. (1999) J. Biol. Chem. 274, in press). In this study, we examined the effect of extracellular free Man on biosynthesis of KDN in mouse melanoma B16 and African green monkey kidney COS-7 cell lines. The following new findings are reported. First, the levels of free and bound forms of KDN increased when the cells were cultured in the presence of 20 mM Man. The level of intracellular free KDN in COS-7 and B16 cells increased 47- and 66-fold respectively, compared with the levels in control cells. Second, the elevated expression of free KDN was proportional to the intracellular concentration of free Man. Third, KDN 9-phosphate (KDN-9-P) synthase, which condenses Man 6-phosphate and phosphoenolpyruvate (PEP), forming KDN-9-P, was detected in cell lysates from both cell lines. Fourth, the de novo synthesis of KDN in both cell lines in the Man-rich media was unaffected by the addition of N-acetylmannosamine (ManNAc), the hexosamine precursor for synthesis of N-acetylneuraminic acid (Neu5Ac). These results show that KDN is synthesized using free Man as its hexose precursor in these mammalian cells. Thus, the KDN biosynthetic pathway utilizes enzymes distinct, at least in part, from those involved in Neu5Ac biosynthesis. This is the first report showing that in vivo synthesis of KDN can be manipulated by growing cells in the presence of Man. This now provides a useful method to study the metabolism and function of the KDN glycotope.

High Performance Liquid Chromatographic Determination of Leukotrienes

Leukotrienes are biologically active compounds derived from lipoxygenasecatalyzed metabolism of arachidonic acid in mammalian cells and tissues. The present report describes a high performance liquid chromatographic (HPLC) method for separation and quantitation of both the peptide (LTC_4, LTD_4, LTE_4) and non peptide (LTB_4) leukotrienes along with a solid phase extraction method for their analysis in biological samples. The mobile phase in the HPLC system, consisting of acetonitrile: water: methanol: acetic acid (300:420: 100:0.8) (v:v) with pH value adjusted to 5.1, proved to be suitable for optimal separation. The isocratic elutiOn of mix standards, containing LTB_4, LTC_4, LTD_4, LTE_4 and PGB_2 as the internal standard, with a flow rate of 1 ml/min through a C 18 column, provided high resolution and reproducible retention times (3.7%, 0.5%, 0.3%, and 0.8% for LTC_4, LTE_4, LTD_4,and L TB_4 respectively) . The liquid solid extraction procedure applied using C18 cartridges of 300mg yielded satifactorily high recovery values with the standards dissolved in phosphate buffer saline solution as well as in plasma. It is concluded that this method provides efficient extraction and quantitative determinaton of leukotrienes in biological fluids.

Constitutive expression of a saturable transport system for non-esterified fatty acids in Xenopus laevis oocytes

In the presence of 150 microM BSA, uptake of [3H]oleate by Xenopus laevis oocytes was a saturable function of the unbound oleate concentration (Vmax. 110 +/- 4 pmol/h per oocyte; Km 193 +/- 11 nM unbound oleate). Oleate uptake was three orders of magnitude faster than that of another test substance, [35S]bromosulphophthalein, and was competitively inhibited by 55 nM unbound palmitate (Vmax. 111 +/- 14 pmol/h per oocyte; Km 424 +/- 63 nM unbound oleate) (P < 0.01). Oleate uptake was also inhibited by antibodies to a 43 kDa rat liver plasma-membrane fatty acid-binding protein, a putative transporter of long-chain fatty acids in mammalian cells; uptake of the medium-chain fatty acid [14C]octanoate was unaffected. Immunofluorescence and immunoblotting demonstrated that the antiserum reacted with a single 43 kDa protein on the oocyte surface. Hence a protein related to the mammalian plasma-membrane fatty acid-binding protein may play a role in saturable uptake of long-chain fatty acids by Xenopus oocytes.

Expression of a mutant Gi2 alpha subunit inhibits ATP and thrombin stimulation of cytoplasmic phospholipase A2-mediated arachidonic acid release independent of Ca2+ and mitogen-activated protein kinase regulation.

The 85-kDa cytoplasmic phospholipase A2 (cPLA2) is the major hormone and growth factor-regulated enzyme that catalyzes release of arachidonic acid in mammalian cells. Activation of cPLA2 requires elevation of intracellular Ca2+ and the phosphorylation of the cPLA2 enzyme by mitogen-activated protein (MAP) kinase. Down-regulation of protein kinase C by phorbol esters or pertussis toxin catalyzed ADP-ribosylation of Gi proteins inhibits thrombin and ATP receptor-stimulated MAP kinase and arachidonic acid release, indicating that functional protein kinase C and Gi proteins are required for G protein regulation of arachidonic acid release. A mutant G alpha i2 subunit having Gly203 mutated to Thr (alpha i2G203T) inhibited thrombin and ATP receptor stimulation of arachidonic acid release independent of adenylyl cyclase inhibition, Ca2+ mobilization, and MAP kinase activation. Overexpression of the wild-type alpha i2 polypeptide or the inactive mutant alpha i2G204A (Gly204 mutated to Ala) polypeptide had no effect on thrombin or ATP receptor stimulation of arachidonic acid release. The phenotype observed with expression of the mutant alpha i2G203T polypeptide defines a role for Gi2 in the control of cPLA2 activity and subsequent arachidonic acid release in addition to the regulation of intracellular Ca2+ levels and MAP kinase activity.

Vimentin serves as a phosphate sink during the apparent activation of protein kinases by okadaic acid in mammalian cells.

The vimentin contents of four mammalian cell lines originating from rat and human tissues were determined by immunoblotting and scanning densitometry. On per cell volume basis, vimentin content in 9L, KD, and HeLa cells was found to be 206.6, 151.6, and 19.1 ng/microliters, respectively. A431 cells were devoid of vimentin. Protein phosphorylation was augmented by treatment of 600 nM okadaic acid for 1 h in these cells. During the apparent activation of protein kinases, vimentin became hyperphosphorylated and the phosphorylation level of other nonvimentin phosphoproteins was relatively little affected in 9L and KD cells. In contrast, cytokeratins and other nonvimentin proteins were heavily phosphorylated in OA-treated HeLa and A431 cells. Regression analysis indicated that the relative increase in phosphorylation level of nonvimentin phosphoproteins was inversely correlated to the contents of vimentin in the four cell lines [r2 = -0.985]. These observations strongly suggest that vimentin acts as a phosphate sink by which the effects of "excess kinase activity" inflicted by phosphatases inhibition was attenuated.

Effect of the immediate precursors of phenylalanine and tyrosine on growth and protein synthesis in phenylalanine- and tyrosine-deprived HeLa cells

Although Phe is an essential amino acid in mammalian cells, its immediate precursor, β-phenylpyruvic acid (BPP), when present in Phe-deficient medium at 10 −4 and 10 −3 M is converted at a sufficient rate to Phe to sustain growth at 60 and 100% of non-deficient control HeLa S-3 cells, respectively. In contrast, Tyr-deficient cells were unable to convert the immediate precursor of Tyr, OH-β-phenylpyruvic acid (OHBPP), nor could BPP rescue Tyr-deficient cells. The results are considered in terms of the organization of intracellular pathways by which precursors are transaminated and made available for protein synthesis.

Transport of cationic amino acids by the mouse ecotropic retrovirus receptor.

Susceptibility of rodent cells to infection by ecotropic murine leukaemia viruses (MuLV) is determined by binding of the virus envelope to a membrane receptor that has multiple membrane-spanning domains. Cells infected by ecotropic MuLV synthesize envelope protein, gp70, which binds to this receptor, thereby preventing additional infections. The consequences of envelope-MuLV receptor binding for the infected host cell have not been directly determined, partly because the cellular function of the MuLV receptor protein is unknown. Here we report a coincidence in the positions of the first eight putative membrane-spanning domains found in the virus receptor and in two related proteins, the arginine and histidine permeases of Saccharomyces cerevisiae (Fig. 1), but not in any other proteins identified by computer-based sequence comparison of the GenBank data base. Xenopus oocytes injected with receptor-encoding messenger RNA show increased uptake of L-arginine, L-lysine and L-ornithine. The transport properties and the expression pattern of the virus receptor behave in ways previously attributed to y+, the principal transporter of cationic L-amino acids in mammalian cells.

Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase activity.

Homogeneous native and recombinant porcine liver thioltransferase (glutaredoxin), bovine thymus and human placenta thioltransferase (glutaredoxin) were examined for dehydroascorbate reductase activity (EC 1.8.5.1) involving the direct catalytic reduction of dehydroascorbic acid (DHA) by glutathione. Each enzyme had substantial activity with apparent Km and Vmax for dehydroascorbate between 0.2 and 2.2 mM and 6-27 nmol min-1, respectively, and for gluathione between 1.6 and 8.7 mM and 11-30 nmol min-1, respectively. In the presence of purified bovine liver thioredoxin reductase, homogeneous bovine liver thioredoxin failed to reduce DHA to ascorbic acid as measured by NADPH oxidation. Highly purified bovine liver protein disulfide isomerase (PDI) reacted directly with DHA and GSH to catalyze the reduction of DHA to ascorbic acid. The apparent Km for DHA was 1.0 mM and the Vmax was 8 nmol min-1, and for GSH were 3.9 mM and 14 nmol min-1, respectively. These results suggest that thioltransferase and PDI contribute to the regeneration of oxidized ascorbic acid in mammalian cells, and based on their cellular location, thioltransferase is proposed to be the major cytoplasmic activity, whereas interaction of DHA with microsomal membrane PDI may catalyze regeneration of ascorbic acid and initiate oxidation of intralumenal protein thiols to disulfides.

The mutagenic activity of aristolochic acid in mammalian cells in vitro is modified by the oxygen tension in the culture medium

The mutagenic activity of aristolochic acid in mammalian cells in vitro is modified by the oxygen tension in the culture medium

In vitro oxidation of ascorbic acid and its prevention by GSH

The interaction of glutathione (GSH) with ascorbic acid and dehydroascorbic acid was examined in in-vitro experiments in order to examine the role of GSH in protecting against the autoxidation of ascorbic acid and in regenerating ascorbic acid by reaction with dehydroascorbic acid. If a buffered solution (pH 7.4) containing 1.0 mM ascorbic acid was incubated at 37°C, there was a rapid loss of ascorbic acid in the presence of oxygen. When GSH was added to this solution, ascorbic acid did not disappear. Maximum protection against ascorbic acid autoxidation was achieved with as little as 0.1 mM GSH. Cupric ions (0.01 mM) greatly accelerated the rate of autoxidation of ascorbic acid, an effect that was inhibited by 0.1 mM GSH. Other experiments showed that GSH complexes with cupric ions, resulting in a lowering of the amount of GSH in solution as measured in GSH standard curves. These results suggest that the inhibition of ascorbic acid autoxidation by GSH involves complexation with cupric ions that catalyze the reaction. When ascorbic acid was allowed to autoxidize at 37°C the subsequent addition of GSH (up to 10 mM) did not lead to the regeneration of ascorbic acid. This failure to detect a direct reaction between GSH and the dehydroascorbic acid formed by oxidation of ascorbic acid under this condition was presumably due to the rapid hydrolysis of dehydroascorbic acid. When conditions were chosen, i.e., low temperature, that promote stability of dehydroascorbic acid, the direct reaction between GSH and dehydroascorbic acid to form ascorbic acid was readily detected. The marked instability of dehydroascorbic acid at 37°C raises questions regarding the efficiency of the redox couple between GSH and dehydroascorbic acid in maintaining the concentration of ascorbic acid in mammalian cells exposed to an oxidative challenge.

Regulation and genetics of amino acid transport.

The major transport systems for the uptake of neutral amino acids in mammalian cells have been designated A, ASC, and L.’,’ The transport systems and their regulation have been characterized in Chinese hamster ovary (CHO) System A is sodium-dependent, subject to trans-inhibition, and serves for the uptake of amino acids with short, polar, or linear side chains. System ASC is also sodium dependent and has a strong preference for alanine, serine, and cysteine. In the C H O cell, the ASC system shows a somewhat broader specificity than that found in the Ehrlich cell.* Unlike System A, System ASC does not tolerate N-methylated substrates such as 2-methylaminoisobutyric acid (MeAIB). System L is sodium-independent and serves for the uptake of branched-chain and aromatic amino acids. We operationally define the systems as follows: System A can be represented by the sodium-dependent uptake of 0.2 m M 2-arninoisobutyric acid (AIB) that is inhibited by 25 m M MeAIB; System ASC, the sodium-dependent uptake of 0.2 m M L-alanine that is not inhibited by 25 m M MeAIB; and System L, the sodium-independent uptake of 0.2 m M L-leucine that is inhibited by 10 mM 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH).3 Although the systems have a preferred set of substrates, they do have overlapping specificities. FIGURE 1 shows the contributions of these systems to the uptake of individual amino acids in CHO-K1 cells. This overlap makes the study of transport systems in isolation difficult. The availability of mutations in one or more of the transport systems would greatly facilitate the study of the function and regulation of the transport systems. We are currently combining genetic approaches with kinetic studies using C H O cells because of the relative ease with which mutants can be obtained from these cells. Furthermore, C H O cells can be used to form interspecies hybrids with human cells.’ The hamster-human hybrid cells preferentially segregate the human chromosomes, permitting the assignment of a phenotype to a particular chromosome. In the present study, we have isolated and characterized C H O mutants defective in the regulation of System L6 and mutants with reduced System L transport activity. We have also used hamster-human hybrids to map System L transport activity to human chromosome 20.’

The effect of glyoxalase I on the metabolism of 4,5-dioxovaleric acid

Biosynthesis of 5-aminolevulinic acid in mammalian cells is catalyzed by aminolevulinic acid synthase in a condensation reaction utilizing glycine and succinyl · coenzyme A. An alternate pathway in mammalian cells may involve the biosynthesis of aminolevulinic acid via a transamination reaction in which L-alanine is the amino donor and 4,5-dioxovaleric acid is the acceptor. This transamination reaction, or one very similar, is employed by plants for the biosynthesis of aminolevulinic acid which is ultimately converted to chlorophyll. The effect of glyoxalase I on the diversion of dioxovaleric acid to other products was tested using both purified glyoxalase I and crude tissue homogenates. Glyoxalase I is a metalloenzyme and glutathione is a co-substrate. Purified glyoxalase I reduced the amount of aminolevulinic acid formed in the presence of dioxovaleric acid, L-alanine, glutathione, and purified L-alanine: 4,5-dioxovaleric acid aminotransferase (dioxovalerate transaminase). The conversion of dioxovaleric acid to aminolevulinic acid was inhibited by the addition of glutathione when a dialyzed bovine liver homogenate served as the source of both glyoxalase I and dioxovalerate transaminase. Removal of metals from bovine liver homogenates produced an 85% decrease in glyoxalase I activity. These ‘metal-free’ homogenates still affected the conversion of dioxovaleric acid to aminolevulinic acid after preincubation with MgSO 4. The effect of glyoxalase I on the metabolism of dioxovaleric acid was also studied using a fluorometric enzyme assay for the quantification of dioxovaleric acid via a coupled enzyme reaction converting it to uroporphyrin. Homogenates of both liver and barley diminished the amount of dioxovaleric acid detected by the coupled assay, but this effect could be prevented by dialysis of the homogenates. Addition of glutathione to dialyzed homogenates markedly reduced the amount of uroporphyrin generated from dioxovaleric acid. Metal-free homogenates supplemented with glutathione reduced the conversion of dioxovaleric acid to uroporphyrin in the coupled assay, but preincubation with MgSO 4 greatly augmented this effect. These studies point out the difficulty in evaluating dioxovaleric acid as a heme precursor using whole cell homogenates.

Purification and characterization of rat kidney l-alanine:4,5-dioxovalerate transaminase and inhibition by hemin

Rat kidney l-alanine:4,5-dioxovalerate transaminase (EC 2.6.1.43), which may be involved in the formation of aminolevulinic acid in mammalian cells, was purified 82-fold to apparent homogeneity with a 19% yield. Molecular weight of the enzyme, as estimated by gel filtration, was found to be 225 000. In polyacrylamide gel electrophoresis under denaturing conditions, the enzyme moved as a single band corresponding to an M r of 37 000, suggesting that the enzyme is composed of six identical subunits. The K m values of l-alanine and 4,5-dioxovalerate are 2.9 and 0.25 mM, respectively. The enzyme had an optimum activity at pH 6.6 and was most active at 65°C. Among some amino acids tested, l-alanine proved to be the most efficient amino donor, and the enzyme was also stereospecific for the l-isomer. The effect of intermediate metabolites of heme biosynthesis, for example, δ-aminolevulinic acid, protoporphyrin, hemin and bilirubin has been studied on purified l-alanine: 4,5-dioxovalerate transaminase. Amongst these metabolites, hemin and protoporphyrin were found to be effective inhibitors.

Leukotrienes: biosynthesis, metabolism, and analysis.

Publisher Summary This chapter discusses biosynthesis, metabolism, and analysis of leukotrienes (LTs). The discovery of the LTs further emphasized the complexity of the metabolism of arachidonic acid in mammalian cells. As the initial report of the occurrence of 5-HETE in rabbit peritoneal polymorphonuclear leukocyte, an impressive number of novel structures has been described and their mechanisms of formation have been elucidated in most cases. Chemical synthesis has made possible pharmacological studies on the LTs, which show potent biological activities, and assay methods are at present available. The presence in a single cell type of several metabolic pathways of arachidonic acid suggests complex regulation mechanisms for the synthesis of the various bioactive metabolites. Although their potent pharmacological properties have been clearly demonstrated, only the measurement of the synthesis of these substances under normal and various pathological situations will permit their regulatory functions in the body to be ascertained. Such studies will require a better knowledge of the in vivo metabolism of the LTs and the development of assay methods for stable plasma or urinary metabolites. Thus, it appears that a new field of biomedical investigation has just been opened.

The role of prostaglandins in the central nervous system.

The prostaglandin system is implicated in physiological and pathological responses of most tissues of the body. The background and breadth of this subject are documented in many reviews and recent symposia (15,25,27, 28,51, 108, 112, 114, 135). This review on the eNS covers advances in the past three years and concerns specifically the possible involvement of the prostaglandin system in the regulation of physiological and pathophysiolog­ ical processes. A wide range of stimuli (hormones, enzymes, trauma, infiammation, pyrogens, immune and allergic reactions, etc) activate a plasma membrane enzyme sequence in mamm alian cells that leads to the rapid de novo synthe­ sis of several prostaglandin types and in certain tissues thromboxanes as well. The biologically active compounds do not accumulate intracellularly and, therefore, under physiological conditions they occur only in trace amounts in tissues and most body fluids. Following formation, action and release, the compounds are rapidly converted by several enzymatic se­ quences to less active or inactive metabolites, which appear in blood and urine. Arachidonic acid, the predominant precursor unsaturated fatty acid in mammalian cells, becomes oxygenated to the prostaglandin endoperox-

Methionine transport in S37 cells. Substrate-dependent function of amino acid transport system A in exchange processes

Methionine had been observed to interact with two principal transport systems for amino acids in mammalian cells, the A and L systems. The present study of methionine transport and of exchange processes through system A arose in the course of a study to define the specificity of a transinhibition effect caused by cysteine. Methionine uptake through two transport systems in the S37 cell was confirmed by the occurrence of a biphasic double-reciprocal plot for labeled methionine uptake. Preloading cells with methionine stimulated labeled histidine uptake through both systems A and L. Efflux of labeled methionine from cells was stimulated by histidine in a biphasic manner, so that both systems A and L can be used for exchange when methionine is the intracellular amino acid. Aminocycloheptanecarboxylic acid elicited exchange efflux of labeled methionine only through system L. α-Aminoisobutyric acid and N- methyl-α- aminoisobutyric acid both stimulated efflux of labeled N- methyl-α- aminoisobutyric acid from S37 cells. These findings are interpreted a showing that transport system A is capable of functioning as an exchange system depending upon the identity of intracellular and extracellular substrates available.