The 4F2 antigen heavy chain induces uptake of neutral and dibasic amino acids in Xenopus oocytes.

The 4F2 cell surface antigen is a disulfide-linked heterodimer induced during the process of cellular activation and expressed widely in mammalian tissues (Parmacek, M. S., Karpinski, B. A., Gottesdiener, K. M., Thompson, C. B., and Leiden, J. M. (1989) Nucleic Acids Res. 17, 1915-1931). The human heavy chain component, a type II membrane glycoprotein, has 29% identity to the amino acid transport-related protein encoded by the recently cloned rat D2 cDNA. We have demonstrated that Xenopus oocytes injected with in vitro transcribed cRNA from D2 take up cystine and dibasic and neutral amino acids (Wells, R. G., and Hediger, M. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5596-5600). In the present study, we examine the role of the human 4F2 heavy chain in amino acid transport. In vitro transcribed 4F2 cRNA was injected into Xenopus oocytes which were assayed for the uptake of radiolabeled amino acids. Our results show that cRNA from 4F2 stimulates the uptake of dibasic and neutral amino acids into oocytes at levels up to 3-fold higher than for water-injected control oocytes. There is no demonstrable uptake of cystine. Uptake is saturable, with characteristics of high affinity transport, and inhibition data suggest that uptake occurs via a single transporter. Dibasic amino acids are taken up by both 4F2 and D2 cRNA-injected oocytes in a sodium-independent manner. In contrast, 4F2-induced but not D2-induced neutral amino acid uptake has a significant component of sodium dependence. Likewise, neutral amino acids in excess inhibit the 4F2-induced uptake of radiolabeled arginine but not leucine in a sodium-dependent manner. The 4F2-induced uptake we observe most likely represents the activity of a single transport system with some characteristics of systems y+, b0,+, and B0,+. We suggest that 4F2 and D2 represent a new family of proteins which induce amino acid transport with distinct characteristics, possibly functioning as transport activators or regulators.     

Functional expression of the intestinal peptide-proton co-transporter in Xenopus laevis oocytes.

The expression of the intestinal peptide-proton cotransporter was examined in Xenopus laevis oocytes by microinjection of poly(A)+ mRNA prepared from rabbit intestinal mucosal cells. The concomitant expression of the glucose-sodium co-transporter was used as the control for the effectiveness of the expression technique. There was significant endogenous activity of Gly-Sar uptake in water-injected oocytes, but the uptake activity increased nearly 3-fold in poly(A)+ mRNA-injected oocytes. The expression of the peptide transporter was time-dependent. There was no detectable expression on day 1 after injection. The expression became noticeable on day 2 and increased with time, reaching a maximum on day 4. There was no further change on days 5 and 6. The endogenous uptake rate measured in water-injected oocytes, on the contrary, showed a slight decrease during this time. The expressed peptide transporter retained its substrate specificity, having affinity for the dipeptides, Gly-Sar and Gly-Pro, and no or little affinity for the free amino acids, Gly and Sar. The expressed peptide transporter also showed a dependence on a transmembrane H+ gradient for maximal activity. These data demonstrate that the mammalian intestinal peptide-proton co-transporter can be successfully expressed in Xenopus laevis oocytes. This expression system can provide an effective assay procedure to clone the gene encoding the transporter.     

DEVELOPMENTAL AND OTHER ASPECTS OF [35S]CYSTEINE TRANSPORT BY RAT BRAIN SYNAPTOSOMES

Abstract— Cysteine uptake by rat brain synaptosomes occurs by active transport. The uptake by synaptosomes isolated from newborn brain is slower and the concentration gradient achieved is lower than that observed in adult tissue. Synaptosomal fractions from both adult and newborn rat brains accumulate cysteine by two saturable systems. The calculated parameters show that the maximum rates of cysteine uptake in adult synaptosomes are approximately twice that observed in newborn synaptosomes for both the high and low affinity systems. The uptake by the high affinity system is sodium dependent and is inhibited by glycine and dibasic amino acids. Uptake by synaptosomes from 14-day-old animals is close to that observed in adult tissue. The uptake of cysteine differs greatly from that of cystine since the oxidized form, cystine, is taken up more slowly by systems with low affinities which are sodium independent, do not interact with dibasic amino acids and are independent of age.     

Cysteine and Cystine Transport at the Blood-Brain Barrier

Abstract: The nature of cysteine and cystine uptake from the cerebral capillary lumen was studied in the rat using the carotid injection technique. [³⁵S]-Cysteine uptake was readily inhibited by the synthetic amino acid 2-amino-bicyclo(2,2,1)heptane-2-carboxylic acid (BCH), the defining substrate for the leucine-preferring (L) system in the Ehrlich ascites cell. The addition of non-radioactive alanine or serine, representatives of the alanine, serine, and cysteine-preferring (ASC) system, produced no significant decrease in the uptake of cysteine after cysteine transport by the L system was blocked with BCH. This indicated that the major component of cysteine's transport from the brain capillary lumen was by the L system with no detectable uptake of cysteine by the ASC system. No carrier-mediated transport of cystine, the disulfide form of the amino acid, was detected, nor was there any inhibition by cystine of the transport of the neutral amino acid methionine or the basic amino acid arginine. These results suggest that the ASC system, if present, is not quantitatively important for the transport of neutral amino acids from the brain capillary lumen.     

Chinese hamster ovary mRNA-dependent, Na(+)-independent L-leucine transport in Xenopus laevis oocytes.

In freshly prepared uninjected folliculated oocytes, Na(+)-independent leucine uptake is mediated predominantly by a system L-like transport system. Removal of follicular cells, however, results in an irreversible loss of this transport activity. When total poly(A)+ mRNA derived from Chinese hamster ovary (CHO) cells was injected into prophase-arrested stage V or VI Xenopus laevis oocytes, enhanced expression of Na(+)-independent leucine transport was observed. The injected mRNAs associated with increased levels of leucine uptake were between 2 and 3 kb in length. The newly expressed leucine transport activity exhibited important differences from the known characteristics of system L, which is the dominant Na(+)-independent leucine transporter in CHO cells as well as in freshly isolated folliculated oocytes. The CHO mRNA-dependent leucine uptake in oocytes was highly sensitive to the cationic amino acids lysine, arginine, and and ornithine (> 95% inhibition). As with the leucine uptake, an enhanced lysine uptake was also observed in size-fractionated CHO mRNA-injected oocytes. The uptakes of leucine and lysine were mutually inhibitable, suggesting that the newly expressed transporter was responsible for uptakes of both leucine and lysine. The inhibition of uptake of lysine by leucine was Na+ independent, thus clearly distinguishing it from the previously reported endogenous system y+ activity. Furthermore, the high sensitivity to tryptophan of the CHO mRNA-dependent leucine transport was in sharp contrast to the properties of the recently cloned leucine transport-associated gene from rat kidney tissue, although leucine transport from both sources was sensitive to cationic amino acids. Our results suggest that there may be a family of leucine transporters operative in different tissues and possibly under different conditions.     

Expression of the activity of cystine/glutamate exchange transporter,system x(c)(-), by xCT and rBAT

The expression of the activity of cystine/glutamate exchange transporter, designated system x(c)(-), requires two components, xCT and 4F2 heavy chain (4F2hc) in Xenopus oocytes. rBAT (related to b(0,+) amino acid transporter) has a significant homology to 4F2hc and is known to be located in the apical membrane of epithelial cells. To determine whether xCT can associate with rBAT and express the activity of system x(c)(-), xCT, and rBAT were co-expressed in Xenopus oocytes and in mammalian cultured cells. In the oocytes injected with rBAT cRNA alone, the activities of cystine and arginine transport were induced, indicating that the system b(0,+)-like transporter was expressed by associating the exogenous rBAT with an endogenous b(0,+)AT-like factor as reported previously. In the oocytes injected with xCT and rBAT cRNAs, the activity of cystine transport was further induced. This induced activity of cystine transport was partially inhibited by glutamate or arginine and completely inhibited by adding both amino acids. In these oocytes, the activity of glutamate transport was also induced and it was strongly inhibited by cystine. In NIH3T3 cells transfected with xCT cDNA alone, the activity of cystine transport was significantly increased, and in the cells transfected with both xCT and rBAT cDNAs, the activity of cystine transport was further enhanced. The enhanced activity was Na(+)-independent and was inhibited by glutamate and homocysteate. These results indicate that rBAT can replace 4F2hc in the expression of the activity of system x(c)(-) and suggest that system x(c)(-) activity could be expressed in the apical membrane of epithelial cells.     

Expression of rat liver canalicular sulfate carrier in Xenopus laevis oocytes

Poly(A)+ RNA (mRNA)extracted from rat liver was injected into Xenopus laevis oocytes and the expression of sulfate transport was determined by measuring [35S] sulfate uptake. Compared to water-injected oocytes, which exhibited virtually no sulfate uptake, injection of rat liver mRNA resulted in a time- and dose-dependent increase in uptake of sulfate. Depending on the method used for the isolation of the mRNA, sulfate uptake was stimulated after injection (40 ng after 6 days) between 8- and 72-fold compared to water-injected oocytes. Sulfate uptake of oocytes injected with mRNA was found to be sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (IC50 less than 20 microM) and could also be inhibited by thiosulfate. Sulfate uptake of injected oocytes showed Michaelis-Menten kinetics (apparent Km, 0.31 mM) which is similar to the Km of the sulfate/bicarbonate antiporter of rat liver canalicular plasma membranes. After fractionation by a sucrose density gradient, the mRNA encoding for the expressed rat liver sulfate carrier was found in fractions containing messages of 3.5-4.0 kilobases in length.     

Diverse transporters for neutral amino acids in Ricinus communis L. seedlings

Uptake mechanisms for neutral amino acids were investigated by expression of mRNA isolated from seedlings of Ricinus communis L. in Xenopus laevis oocytes. After injection of mRNA from root, hypocotyl and cotyledon currents elicited by saccharose and neutral amino acids ranged from 0.3 nA up to 2 nA depending on the respective substrate and the source of mRNA. These currents were due to expression of low affinity uptake mechanisms and the KM values found for amino acid induced charge flow range from 1 to 2 mM. The abundance and/or the specificity of the expressed mechanisms differ in the various tissues. Currents of similar magnitude were recorded for alanine and glutamine with mRNA isolated from root, hypocotyl and cotyledons. Serine and proline induced currents after injection of mRNA from hypocotyl and roots, in case of α-aminoisobutyric acid (AIB) induced currents were generally small with mRNA from all tissues tested. In addition, differential sensitivity of glutamine and AIB uptake in the high affinity range was evident towards the amino acid analogue 2-chloro-aminophenoxybutyric acid which indicated an additional set of carriers operating in the micromolar concentration range. The results suggest that multiple transporters for neutral amino acids exist in various tissues of the plant differing in specificity of charge flow and in sensitivity towards the inhibitor 2-chloro-aminophenoxybutyric acid. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of rat liver Na+/L-alanine co-transport in Xenopus laevis oocytes. Effect of glucagon in vivo.

Poly(A)+ RNA (mRNA) isolated from rat liver was injected into Xenopus laevis oocytes, and expression of Na+/L-alanine transport was assayed by measuring Na(+)-dependent uptake of L-[3H]alanine. Expression of Na+/L-alanine transport was detected 3-7 days after mRNA injection, and was due to an increment of the Na(+)-dependent component. After injection of 40 ng of total mRNA, Na(+)-dependent uptake of L-alanine was 2.5-fold higher than in water-injected oocytes. In contrast with Na+/L-alanine transport by water-injected oocytes, expressed Na+/L-alanine transport was inhibited by N-methylaminoisobutyric acid, was inhibited by an extracellular pH of 6.5 and was saturated at approx. 1 mM-L-alanine. After sucrose-density-gradient fractionation, highest expression of Na+/L-alanine uptake was observed with mRNA of 1.9-2.5 kb in length. Compared with mRNA isolated from control rats, mRNA isolated from glucagon-treated rats showed a approx. 2-fold higher expression of Na+/L-alanine transport. The results demonstrate that both liver Na+/L-alanine transport systems (A and ASC) can be expressed in X. laevis oocytes. Furthermore, the data obtained with mRNA isolated from glucagon-treated rats suggest that glucagon regulates liver Na+/L-alanine transport (at least in part) via the availability of the corresponding mRNA.     

Characteristics of cystine uptake by cultured LLC-PK1 cells

The characteristics of the uptake of L-cystine by LLC-PK1 cells were examined. The uptake diminished with time in culture after passage of cells while the uptake of sugar increased. In 48-h-cultured cells at a range of cystine concentrations including physiological levels uptake occurred via a saturable process which was independent of medium sodium concentration and pH. No inhibition of cystine uptake occurred in the presence of lysine which is known to share the cystine transport system in uncultured renal proximal tubule cells and brush-border membrane vesicles. Glutamate was a potent inhibitor of cystine uptake and participated in heteroexchange diffusion with cystine. The cystine-glutamate transport process resembles that of cultured human fibroblasts. The inability of these cells to reflect the genetically determined cystine-lysine system which is altered in the kidney in human cystinuria makes them an inappropriate model of the renal tubule cell cystine transport system. On the other hand, they may provide a model system for examining the factors which determine the presence of the various cystine transport process.     

l-[35S]CYSTINE UPTAKE BY RAT BRAIN SYNAPTOSOMES

Abstract— The uptake of [³⁵S]cystine at 37°C by synaptosomal fractions isolated from adult rat cerebrum can be divided into two components. About 60% of the uptake is due to binding to synaptosomal proteins while the remainder exists as a free amino acid pool. Chemical analysis of this soluble component indicates that considerable reduction of cystine to cysteine occurs with 75% or more of the labeled molecular species being cysteine. The process involved in the uptake into the soluble pool was composed of two saturable systems with apparent K _m values of 0.14 and 1.4 m m . The low K _m system was sodium and oxygen independent but inhibited by dinitrophenol. Dibasic amino acids, lysine, arginine and ornithine, did not inhibit cystine uptake. The characteristics of cystine uptake by synaptosomes from newborn brain are very similar to those of adult brain.     

Expression of Size-Selected RNA Encoding Brain Serotonin Transporter in Xenopus laevis Oocytes

The mRNA that encodes a serotonin transporter was expressed using the Xenopus laevis oocyte expression system. Poly(A)+ RNA isolated from mouse brainstem was injected into Xenopus laevis oocytes, and the ability of oocytes to take up serotonin was measured 3 days postinjection. RNA-dependent serotonin uptake was sensitive to citalopram, a specific inhibitor of serotonin uptake, whereas background levels of serotonin uptake were not citalopram sensitive. Two RNA size fractions, 4.0 and 4.5 kb, were most efficient in stimulating uptake. Injection into Xenopus laevis oocytes of the 4.5-kb size fraction of mouse brainstem RNA resulted in threefold more serotonin uptake than did injection of unfractionated poly(A)+ RNA.     

Mouse lymphoma L1210 cells acquire a new cystine transport activity upon adaptation in vitro

Summary Mouse lymphoma L1210 cells maintained in vitro at a high cell density for a certain time period adapted themselves to the in vitro environment and were able to grow indefinitely. From these adapted cells, more than 30 clones were isolated. They all had much higher activity to take up cystine than the original L1210 cells, supporting a previous view that the deficiency of the cystine uptake limits the survival and growth of L1210 cells in vitro. The cystine uptake of one cloned cell line was characterized. The enhanced uptake of cystine in these cells was mainly mediated by a Na⁺-independent, saturable system and was potently inhibited by glutamate and some other anionic amino acids, but less by aspartate. Such activity of cystine uptake was not observed in the original L1210 cells. The results suggest that, upon adaptation in vitro, L1210 cells acquire a new cystine transport activity necessary for survival and growth in vitro.     

Expression of phenolic and tyrosyl ring iodothyronine deiodinases in Xenopus laevis oocytes is dependent on the tissue source of injected poly(A)+ RNA

The phenolic (5' position) and tyrosyl (5 position) ring deiodinases which catalyze the peripheral metabolism of thyroid hormones have proven difficult to purify and characterize biochemically. The present studies used Xenopus laevis oocytes as an in vivo translational assay system for detecting and quantitating mRNA for these enzymes. The injection of poly(A)+ RNA prepared from a human term placenta induced 5-deiodinase activity in oocytes. The expressed activity increased for up to 96 h after injection, was proportional to the amount of RNA injected, and manifested a Michaelis-Menten constant (Km) for T3 of 1.6 nM. In oocytes injected with poly(A)+ RNA prepared from rat liver, anterior pituitary gland, or brown adipose tissue, 5-deiodinase activity could not be demonstrated. The injection of poly(A)+ RNA from 15-day-old chick embryonic liver induced both 5'- and 5-deiodinase activity, with the 5'-deiodinase activity being sensitive to inhibition by 6-n-propyl-2-thiouracil. X. laevis oocytes can thus be induced to express either phenolic or tyrosyl ring deiodinase activity, or both, by the microinjection of poly(A)+ RNA prepared from selected tissues. These findings demonstrate that the types of deiodinase activity present in different organs represent tissue specific patterns of mRNA expression and strongly suggest that the enzymes responsible for types I and III deiodinase activity are encoded by different mRNAs.     

Expression of Madin-Darby canine kidney cell Na(+)-and Cl(-)-dependent taurine transporter in Xenopus laevis oocytes.

Expression of a Madin-Darby canine kidney (MDCK) cell taurine transporter was examined in Xenopus oocytes that had been injected with poly(A)+ RNA extracted from MDCK cells. Compared with water-injected oocytes, injection of total poly(A)+ RNA resulted in an increase in Na(+)-dependent taurine uptake which was directly related to the amount of RNA injected. The magnitude of expression in poly(A)+ RNA-injected oocytes was 5-10-fold higher than that of water-injected oocytes. Since the Vmax of taurine uptake in MDCK cells is increased by culture in hypertonic medium, we compared oocyte taurine uptake after injection with poly(A)+ RNA from MDCK cells cultured in hypertonic medium with uptake in oocytes injected with poly(A)+ RNA from hypertonic cells elicited twice the taurine uptake elicited by poly(A)+ RNA from isotonic cells. The transporter expressed in oocytes was like that in MDCK cells: it was completely dependent on external sodium and was also anion dependent (Cl- greater than or equal to Br- greater than SCN- much greater than gluconate-). Other beta-amino acids, beta-alanine and hypotaurine, inhibited taurine uptake, but L-alanine and 2-(methylamino) isobutyric acid did not. The apparent Km of the transporter was 7.0 microM. After size fractionation on a sucrose density gradient, poly(A)+ RNA encoding for the MDCK taurine transporter was found in the fraction whose average size was 4.4 kilobases.     

The influence of pH on cystine and dibasic amino acid transport by rat renal brushborder membrane vesicles

The uptake of cystine and lysine by rat renal brushborder membrane vesicles was examined at various intravesicular and extravesicular hydrogen ion concentrations to discern whether ionic species are determinants of specificity for the shared transport system and whether hydrogen ion gradients play a role in determining uptake values. When intravesicular and extravesicular pH are identical, the highest uptake of cystine occurred at pH 7.4, with lesser uptake at pH 6.0 and 8.3. Since cystine is electroneutral at pH 6.0 and 90% anionic at pH 8.3, it appears that neither form of the amino acid is a preferred species for transport. A similar relationship between pH and uptake occurs for lysine, which is cationic at pH below 8.5. This suggests that pH affects the functioning of the membrane carrier system independent of ionic species of the substrate. There is no apparent relationship of cystine uptake to hydrogen ion gradients across the membrane. Over the range of extravesicular pH studied, optimal cystine uptake occurred whenever the intravesicular pH was 7.4. Competitive interactions between unlabeled amino acids and labeled cystine were not affected by the extravesicular pH and, therefore, did not seem determined by the ionic species of cystine.     

The molecular basis of cystinuria: the role of the rBAT gene

Summary The cDNAs of mammalian amino acid transporters already identified could be grouped into four families. One of these protein families is composed of the protein rBAT and the heavy chain of the cell surface antigen 4F2 (4F2hc). The cRNAs of rBAT and 4F2hc induce amino acid transport activity via systems b^0,+ -like and y⁺L -like inXenopus oocytes respectively. Surprisingly, neither rBAT nor 4F2hc is very hydrophobic, and they seem to be unable to form a pore in the plasma membrane. This prompted the hypothesis that rBAT and 4F2hc are subunits or modulators of the corresponding amino acid transporters. The association of rBAT with a light subunit of ~40kDa has been suggested, and such an association has been demonstrated for 4F2hc.The b^0,+-like system expressed in oocytes by rBAT cRNA transports L-cystine, L-dibasic and L-neutral amino acids with high-affinity. This transport system shows exchange of amino acids through the plasma membrane ofXenopus oocytes, suggesting a tertiary active transport mechanism. The rBAT gene is mainly expressed in the outer stripe of the outer medulla of the kidney and in the mucosa of the small intestine. The protein localizes to the microvilli of the proximal straight tubules (S3 segment) of the nephron and the mucosa of the small intestine. All this suggested the participation of rBAT in a high-affinity reabsorption system of cystine and dibasic amino acids in kidney and intestine, and indicated rBAT (named SLC3A1 in Gene Data Bank) as a good candidate gene for cystinuria. This is an inherited aminoaciduria due to defective renal and intestinal reabsorption of cystine and dibasic amino acids. The poor solubility of cystine causes the formation of renal cystine calculi. Mutational analysis of the rBAT gene of patients with cystinuria is revealing a growing number (~20) of cystinuria-specific mutations, including missense, nonsense, deletions and insertions. Mutations M467T (substitution of methionine 467 residue for threonine) and R270X (stop codon at arginine residue 270) represent approximately half of the cystinuric chromosomes where mutations have been found. Mutation M467T reduces transport activity of rBAT in oocytes. All this demonstrates that mutations in the rBAT gene cause cystinuria.Three types of cystinuria (types, I, II and III) have been described on the basis of the genetic, biochemical and clinical manifestations of the disease. Type I cystinuria has a complete recessive inheritance; type I heterozygotes are totally silent. In contrast, type II and III heterozygotes show, respectively, high or moderate hyperaminoaciduria of cystine and dibasic amino acids. Type III homozygotes show moderate, if any, alteration of intestinal absorption of cystine and dibasic amino acids; type II homozygotes clearly show defective intestinal absorption of these amino acids. To date, all the rBAT cystinuria-specific mutations we have found are associated with type I cystinuria (~70% of the chromosomes studied) but not to types II or III. This strongly suggests genetic heterogeneity for cystinuria. Genetic linkage analysis with markers of the genomic region of rBAT in chromosome 2 (G band 2p16.3) and intragenic markers of rBAT have demonstrated genetic heterogeneity for cystinuria; the rBAT gene is linked to type I cystinuria, but not to type III. Biochemical, genetic and clinical studies are needed to identify the additional cystinuria genes; a low-affinity cystine reabsortion system and the putative light subunit of rBAT are additional candidate genes for cystinuria.     

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.     

Absence of a role of gamma-glutamyl transpeptidase in the transport of amino acids by rat renal brushborder membrane vesicles

Summary The role of the enzyme, gamma-glutamyl transpeptidase on the uptake of amino acids by the brushborder membrane of the rat proximal tubule was examined by inhibiting it with AT-125 (l-[S, 5S]--amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). AT-125 inhibited 98% of the activity of gamma-glutamyl transpeptidase when incubated for 20 min at 37°C with rat brushborder membrane vesicles. AT-125 given to ratsin vivo inhibited 90% of the activity of gamma-glutamyl transpeptidase in subsequently isolated brushborder membrane vesicles from these animals. AT-125 inhibition of gamma-glutamyl transpeptidase bothin vivo andin vitro had no effect on the brushborder membrane uptake of cystine. Similarly, there was no effect of gamma-glutamyl transpeptidase inhibition by AT-125 on glutamine, proline, glycine, methionine, leucine or lysine uptake by brushborder membrane vesicles. Furthermore, the uptake of cystine by isolated rat renal cortical tubule fragments, in which the complete gamma-glutamyl cycle is present, was unaffected by AT-125 inhibition of gamma-glutamyl transpeptidase. Therefore, in the two model systems studied, gamma-glutamyl transpeptidase did not appear to play a role in the transport of amino acids by the renal brushborder membrane.