Elucidation of an a and L system for amino acid transport in the human lymphoblast using a membrane filtration technique

Summary Optimum conditions have been established for the measurement of amino acid transport by human lymphoblastoid cell lines using a membrane-filtration technique. The parameters we found to be important for the reproducibility of the method are: the types and combination of filters, the strength of the vacuum applied to the filters and the density of the cultures at the time of harvesting and during uptake and filtration. We found that bovine serum albumin added to phosphate buffered saline (PBS) glucose in which the cells are washed, resuspended and assayed is essential for the maintenance of viability, the prevention of clumping and the retention of the accumulated amino acid. Using this procedure we have characterized two transport systems for the neutral amino acids; an A and an L system, which are similar but not identical to the A and L systems characterized in rodent cell lines. These A and L systems have characteristically lower Km's and Vm's for alanine and phenylalanine, when compared to rodent cell lines. In addition, we find α-AIB to be a poor competitor of alanine and phenylalanine uptake. This work was supported by Grant No. CA18644, awarded by the National Cancer Institute, Department of Health, Education and Welfare, and from a grant from the National Science Foundation under Grant No. PCM 76-24328.     

Carbohydrate Effects on Amino Acid Transport by Trypanosoma equiperdum*

SYNOPSIS. Uptake of ¹⁴C-labeled alanine, glutamate, lysine, methionine, proline, and phenylalanine by Trypanosoma equiperdum during 2-minute incubations occurred by diffusion and membrane-mediated processes. Amino acid metabolism was not detected by paper chromatography of trypanosome extracts. Most of 18 carbohydrates tested for ability to alter amino acid transport neither changed nor significantly inhibited transport. Glucose, however, stimulated glutamate, lysine and proline transport; fructose stimulated lysine uptake and 2-deoxy-D-glucose increased phenylalanine and methionine absorption. No evidence was found that the carbohydrates acted by binding to amino acid transport “sites.” Glucose inhibition of alanine, phenylalanine, and methionine uptake was linked to glycolysis. The rapid formation of alanine from glucose stimulated alanine release and, when glycolysis was blocked, glucose no longer inhibited alanine transport. Methionine and phenylalanine release was also stimulated by glucose. Glucose changed the ability of lysine, glutamate, and proline to inhibit each others’uptake, indicating that certain amino acids are preferentially absorbed by respiring cells. Analysis of free pool amino acid levels suggested that some amino acid transport systems in T. equiperdum are linked in such a way to glycolysis as to control the cell concentrations of these amino acids.     

Amino acid transport in normal and Rous sarcoma virus-transformed chicken embryo fibroblasts.

A study was made of the transport of a variety of amino acids by uninfected and Rous sarcoma virus-infected chicken embryo fibroblasts. Following a period of amino acid starvation, transformed, but not normal cells, showed increased levels of transport for alpha-aminoisobutyric acid, proline and alanine, three amino acids which are transported primarily by the A transport system. There was no starvation-induced increase in the transport of leucine, phenylalanine, lysine, or cycloleucine. In the absence of starvation, normal and transformed cells exhibited comparable rates of amino acid transport. Cycloheximide was able to block the increase in uptake. The enhanced uptake was characterized by an increase in Vmax for transport and little change in Km. The data demonstrate that an alteration in the regulation of the A amino acid transport system is an early event in malignant transformation by Rous sarcoma virus. However, since this alteration in made manifest only following a period of starvation, our findings suggest that increased amino acid uptake does not play a role in generating the other manifestations of the transformed state seen in cell culture.     

Acidic amino acid transport in animal cells and tissues

1. The occurrence and characterization of acidic amino acid transport in the plasma membrane of a variety of cells and tissues of a number of organisms is reviewed. 2. Several cell types, especially in brain, possess both high- and low-affinity transport systems for acidic amino acids. 3. High-affinity systems in brain may function to remove neurotransmitter amino acid from the extracellular environment. 4. Many cell systems for acidic amino acid transport are energized by an inwardly directed Na+ gradient. Moreover, certain cell types, such as rat brain neurons, human placental trophoblast and rabbit and rat kidney cortex epithelium, respond to an outwardly directed K+ gradient as an additional source of energization. This simultaneous action may account for the high accumulation ratios seen with acidic amino acids. 5. Rabbit kidney has been found to have a glutamate-H+ co-transport system which is subject to stimulation by protons in the medium. 6. Acidic amino acid transport in rat brain neurons occurs with a stoichiometric coupling of 1 mol of amino acid to 2 mol of Na+. For rabbit intestine, one Na+ is predicted to migrate for each mol of amino acid. 7. Uptake in rat kidney cortex and in high-K+ dog erythrocytes is electrogenic. However, uptake in rabbit and newt kidney and in rat and rabbit intestine is electroneutral. 8. Na+-independent acidic amino acid transport systems have been described in the mouse lymphocyte, the human fibroblast, the mouse Ehrlich cell and in rat hepatoma cells. 9. In a number of cell systems, D-acidic amino acids have substantial affinity for transport; D-glutamate, in a number of systems, however, appears to have little reactivity. 10. Acidic amino acid transport in some cell systems appears to occur via the "classical" routes (Christensen, Adv. Enzymol. Relat. Areas Mol. Biol. 49, 41-101, 1979). For example, uptake in the Ehrlich cell is partitioned between the Na+-dependent A system (which transports a wide spectrum of neutral amino acids), the Na+-dependent ASC system (which transports alanine, serine, threonine, homoserine, etc.), and the Na+-independent L system (which shows reactivity centering around neutral amino acids such as leucine and phenylalanine). Also, a minor component of uptake in mouse lymphocytes occurs by a route resembling the A system. 11. Human fibroblasts possess a Na+-independent adaptive transport system for cystine and glutamate that is enhanced in activity by cystine starvation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Growth regulation and amino acid transport in epithelial cells: Influence of culture conditions and transformation on A,ASC, and l transport activities

Amino acid transport in Madin-Darby canine kidney (MDCK) cells, grown in a defined medium, was investigated as a function of cell density, exposure to specific growth factors, and transformation. MDCK cells were found to transport neutral amino acids by systems similar to the A, ASC, L, and N systems which have been characterized using other cell lines. Experimental conditions were developed for MDCK cells which allowed independent measurement of A, ASC, and L transport activities. The activity of the L system was measured as Na⁺-independent leucine or methionine uptake at pH 7.4. The activity of the A system was measured as Na⁺-dependent α(methylamino)isobutyric acid (mAIB) uptake at pH 7.4, the activity of the ASC system was measured as Na⁺-dependent alanine uptake in the presence of 0.1 mM mAIB at pH 6.0, and the activity of system N was observed by measuring Na⁺-dependent glutamine uptake at pH 7.4 in the presence of high concentrations of A and ASC system substrates. The L transport system responded minimally to changes in growth state, but Na⁺-dependent amino add transport responded to regulation by growth factors, cell density, and transformation. The activities of the A and ASC systems both decreased at high cell density, but these activities responded dissimilarly under other conditions. The activity of the A system was stimulated by insulin, was inhibited by PGE₁, and was elevated 3–7 fold in the transformed cell line, MDCK-T₁. The activity of the ASC system was slightly stimulated by insulin and by PGE₁, but was unchanged after chemical transformation. Changes in cellular growth were monitored and were found to correlate best with the activity of the A system. These results suggested that MDCK cell growth may be more closely related to the activity of the A than of the ASC system.     

Maturation of membrane function: transport of amino acid by rat erythroid cells.

The membrane changes which occur during cellular maturation of erythroid cells have been investigated. The transport of alpha-aminoisobutyric acid, alanine, and N-methylated-alpha-aminoisobutyric acid have been studied in the erythroblastic leukemic cell, the reticulocyte, and the erythrocyte of the Long-Evans rat. The dependence of amino acid transport on extracellular sodium concentration was investigated. Erythrocytes were found to transport these amino acids only by Na-independent systems. The steady state distribution ratio was less than 1. Reticulocytes were found to transport alpha-aminoisobutyric acid and alanine by Na-dependent systems, but only small amounts of N-methylated-alpha-aminoisobutyric acid. Small amounts of these amino acids were transported by Na-independent systems. The steady state distribution ratio was greater than one for Na-dependent transport. The erythroblastic leukemia cell, a model immature erythroid cell, showed marked Na-dependence (greater than 90%) for alpha-aminoisobutyric acid and alanine transport, and greater than 80% for the Na-dependent transport of N-methyl-alpha-aminoisobutyric acid. The steady state distribution ratio for the Na-dependent transport was greater than 4. In the erythroblastic leukemic cell, at least three Na-dependent systems are present: one includes alanine and alpha-aminoisobutyric acid, but excludes N-methyl-alpha-aminoisobutyric acid; one is for alpha-aminoisobutyric acid, alanine and also N-methyl-alpha-aminoisobutyric acid; and one is for N-methyl-alpha-aminoisobutyric acid alone. In the reticulocyte, the number of Na-dependent systems are reduced to two: one for alpha-aminoisobutyric acid and alanine; one for N-methyl-alpha-aminoisobutyric acid. In the erythrocytes, no Na-dependent transport was found. Therefore, maturation of the blast cell to the mature erythrocyte is characterized by a systematic loss in the specificity and number of transport system for amino acids.     

Transport of amino acids in intact 3T3 and SV3T3 cells. Binding activity for leucine in membrane preparations of ehrlich ascites tumor cells

Transport of amino acids into 3T3 and SV3T3 (SV40 virus-transformed 3T3) cells was measured on glass cover slips. The 3T3 and SV3T3 cells contain both A (alanine preferring) and L (leucine preferring) systems for neutral amino acid transport. Initial rates of uptake of amino acids are about twofold higher in SV3T3 than in 3T3 cells. Other parameters measured, however, do not indicate marked differences in the transport of amino acids by the two cell types. L-system amino acids, such as leucine, are subject to trans-stimulation in both cell lines, whereas A-system amino acids, such as alanine and glycine, are not. Leucine was transported to higher levels in confluent cells than in nonconfluent cells. Glycine, however, shows distinctly less transport activity as the cells become confluent. Ehrlich ascites cell plasma membranes were prepared and assayed for amino acid-binding activity. Leucine-binding activity was detected by equilibrium dialysis in Triton X-100-treated membrane preparations.     

Maturation of membrane function: Transport of amino acid by rat erythroid cells

The membrane changes which occur during cellular maturation of erythroid cells have been investigated. The transport of α-aminoisobutyric acid, alanine, and N-methylated-α-aminoisobutyric acid have been studied in the erythroblastic leukemic cell, the reticulocyte, and the erythrocyte of the Long-Evans rat. The dependence of amino acid transport on extracellular sodium concentration was investigated. Erythrocytes were found to transport these amino acids only by Na-independent systems. The steady state distribution ratio was less than 1. Reticulocytes were found to transport α-aminoisobutyric acid and alanine by Na-dependent systems, but only small amounts of N-methylated-α-aminoisobutyric acid. Small amounts of these amino acids were transported by Na-independent systems. The steady state distribution ratio was greater than one for Na-dependent transport. The erythroblastic leukemia cell, a model immature erythroid cell, showed marked Na-dependence (>90%) for α-aminoisobutyric acid and alanine transport, and >80% for the Na-dependent transport of N-methyl-α-aminoisobutyric acid. The steady state distribution ratio for the Na-dependent transport was >4. In the erythroblastic leukemic cell, at least three Na-dependent systems are present: one includes alanine and α-aminoisobutyric acid, but excludes N-methyl-α-aminoisobutyric acid; one is for α-aminoisobutyric acid, alanine and also N-methyl-α-aminoisobutyric acid; and one is for N-methyl-α-aminoisobutyric acid alone. In the reticulocyte, the number of Na-dependent systems are reduced to two: one for α-aminoisobutyric acid and alanine; one for N-methyl-α-aminoisobutyric acid. In the erythrocytes, no Na-dependent transport was found. Therefore, maturation of the blast cell to the mature erythrocyte is characterized by a systematic loss in the specificity and number of transport systems for amino acids.     

Inhibition of sodium-independent amino acid transport by dexamethasone in rat hepatoma cells

We studied the uptake of leucine, phenylalanine, and the amino acid analog, 2-aminonorborane-2-carboxylic acid, by rat hepatoma cells in tissue culture. The uptake of these amino acids was partially mediated by a plasma membrane transport system similar to the L agency described in other cell types in that it does not require extracellular sodium and is subject to trans-stimulation. Initial rates of sodium-independent transport of these amino acids were calculated using mathematical transformations of the uptake time course curves. The glucocorticoid dexamethasone inhibits the activity of this transport system; the initial rates of sodium-independent uptake of leucine, phenylalanine, and 2-aminonorborane-2-carboxylic acid are decreased by approximately one-third (average = 30%, n = 19) after incubation of HTC cells with 0.1 microM dexamethasone. This inhibition requires at least 15 h, reaching a maximum at 24 h of exposure of the cells to the hormone. Dexamethasone has an asymmetrical effect on sodium-independent amino acid transport in that exposure of the cells to the hormone does not inhibit the rates of outflow of leucine or phenylalanine from preloaded cells into medium without sodium. Inhibition of uptake is blocked by 0.1 mM cycloheximide and 4 microM actinomycin D, indicating the need for continuous protein synthesis for dexamethasone action. Insulin, which is known to partially reverse the inhibitory effect of dexamethasone on the A amino acid transport system in HTC cells, does not alter the action of dexamethasone on the L system. Previous investigations have demonstrated inhibition by dexamethasone of at least two distinct sodium-dependent amino acid transport activities in HTC cells. The data presented here, showing inhibition by the glucocorticoid of a sodium-independent transport activity, indicate that the effect of the hormone is independent of the energy source of the amino acid transport systems affected.     

In vivo muscle amino acid transport involves two distinct processes

We have tested the hypothesis that transit through the interstitial fluid, rather than across cell membranes, is rate limiting for amino acid uptake from blood into muscle in human subjects. To quantify muscle transmembrane transport of naturally occurring amino acids, we developed a novel 4-pool model that distinguishes between the interstitial and intracellular fluid compartments. Transport kinetics of phenylalanine, leucine, lysine, and alanine were quantified using tracers labeled with stable isotopes. The results indicate that interstitial fluid is a functional compartment insofar as amino acid kinetics are concerned. In the case of leucine and alanine, transit between blood and interstitial fluid was potentially rate limiting for muscle amino acid uptake and release in the postabsorptive state. For example, in the case of leucine, the rate of transport between blood and interstitial fluid compared with the corresponding rate between interstitial fluid and muscle was 247 +/- 36 vs. 610 +/- 95 nmol.min(-1).100 ml leg(-1), respectively (P < 0.05). Our results are consistent with the process of diffusion governing transit from blood to interstitial fluid without selectivity, and of specific amino acid transport systems with varying degrees of efficiency governing transit from interstitial fluid to muscle. These results imply that changes in factors that affect the transit of amino acids from blood through interstitial fluid, such as muscle blood flow or edema, could play a major role in controlling the rate of muscle amino acid uptake.     

Characterization of neutral amino acid uptake by cultured epithelial cells from pig kidney

Two transport systems for neutral amino acids have been characterised in LLC-PK₁ cells. The first, which transport alanine in a sodium-dependent manner, also mediates alanine exchange and is preferentially inhibited by serine, cysteine, and α-amino-n-butyric acid. This system resembles the ASC system in Ehrlich ascites and some other cell types. There is only a small contribution of other systems to alanine uptake. The second, which transports leucine with no requirement for sodium and mediates leucine exchange, is blocked by 2-aminonorbornane-2-carboxylic acid and hydrophobic amino acids. This system is similar to the L system described in other cell types. LLC-PK₁ cells retain several other features implying renal proximal tubule origin; our results thus suggest that these transport systems may be involved in the reabsorption of neutral amino acids by the nephron in vivo.     

Specificity of Amino Acid Transport in Trypanosoma equiperdum*

Uptake of [¹⁴C] alanine, arginine, glutamic acid and phenylalanine by Trypanosoma equiperdum occurred by both a mediated mechanism and diffusion. Twenty amino acids were studied as inhibitors of absorption of the above amino acids. Results suggested that at least 4 distinct transport loci are involved in amino acid transport. These 4 loci have overlapping affinities for amino acids and seem to be involved, respectively, in the absorption of (a) arginine and phenylalanine; (b) arginine; (c) alanine, phenylalanine, and glutamic acid; (d) glutamic acid. The data also showed that multiple sites for substrate binding occur on each of 2 transport systems.     

INHIBITION PATTERN BY ANALOGS INDICATES THE PRESENCE OF TEN OR MORE TRANSPORT SYSTEMS FOR AMINO ACIDS IN BRAIN CELLS

Abstract— We surveyed the transport systems present in the brain for amino acids. Cellular transport was measured in brain slices, and capillary transport was estimated by measuring in vivo the short-term (15 s) extraction by brain from the blood. Specific analog inhibition of uptake was used to distinguish the classes. Amino acid levels (close to physiological) were such that primarily the 'low-affinity' transport was measured.
In brain tissue we could distinguish 10 overlapping amino acid transport classes. Five of these, described in a number of tissues, were characterized by their substrates: alanine (A system), leucine (L system), alanine-serine-cysteine (ASC system), glutamic acid (Glu system), and arginine (Ly⁺ system), respectively. The others distinguished were each fairly specific for one of the following five amino acids: glycine, proline, γ-aminobutyric acid (GABA), taurine, and lysine. Of these 10 systems only 4 could be clearly found in capillary transport: L, ASC, Ly ⁺, and Glu.
The properties and the distribution of the transport systems are different. Examples are that at least one of the systems is present primarily only in neurons (GABA), and one primarily in glia (taurine). The specificity of some of the systems, e.g. A, is altered during development. In contrast to the properties of most other systems, there is little Na⁺, energy, or temperature dependence of the L system. This is reflected in the properties of capillary neutral amino acid transport when the L system is the predominant one.     

Neutral amino acid transport in human synovial cells: substrate specificity of adaptative regulation and transinhibition

Neutral amino acid transport was characterized in human synovial cells. The amino acids tested are transported by all three major neutral amino acid transport systems, that is, A, L, and ASC. The model amino acid 2-aminoisobutyric acid (AIB) was found to be a strong specific substrate for system A in synovial cells. When cells were starved of amino acids, the activity of AIB transport increased, reaching a maximum within 1 h. The stimulation of transport activity was not blocked by cycloheximide and would thus appear to be related to a release from transinhibition. Similarly, the decrease in the activity of AIB transport observed after the addition of alpha-methyl-aminoisobutyric acid (meAIB) appeared to be related to transinhibition. However, using a different approach, that is, amino acid starvation followed by incubation with 10 mM meAIB and transfer to an amino acid-free medium with or without cycloheximide supplementation, a clear increase in AIB uptake, due both to derepression and a release from transinhibition, was observed. Unlike human fibroblasts, the depression of system A in these synovial cells was not serum-dependent. The process of derepression was observed only after preloading with meAIB. Neither AIB nor alanine produced this phenomenon. Moreover, alanine preloading led to a large increase in AIB transport activity due to a release from transinhibition. These observations indicate that the process of derepression and release from transinhibition are specific to the substrates present in the culture medium prior to amino acid starvation.     

Normal amino acid uptake by cultured human fibroblasts does not require gamma-glutamyl transpeptidase

The uptake kinetics for four amino acids (cystine, glutamine, methionine, and alanine) which are among the best gamma-glutamyl acceptors have been determined for normal human fibroblasts and for a cell line containing undetectable quantities (< 0.5% normal mean) of gamma-glutamyl transpeptidase activity. Apparent Km and V(max) for uptake for each of the four amino acids were normal in the mutant fibroblasts. Insulin increased the uptake of alpha-aminoisobutyrate as in control cells. levels of 16 amino acids were also normal in this cell strain; the intracellular concentrations of phenylalanine, cystine, and cysteine were increased. In human fibroblasts, amino acid transport appears to proceed normally in the absence of active gamma-glutamyl transpeptidase.     

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.     

Transport of L-4-azaleucine in Escherichia coli.

The uptake of L-4-azaleucine was examined in Escherichia coli K-12 strains to determine the systems that serve for its accumulation. L-4=Azaleucine in radio-labeled form was synthesized and resolved by the action of hog kidney N-acylamino-acid amidohydrolase (EC 3.5.1.B) on the racemic alpha-N-acetyl derivative of DL-[dimethyl-14C]4-azaleucine. L-4-Azaleucine is taken up in E. coli by energy-dependent processes that are sensitive to changes in the pH and to inhibition by leucine and the aromatic amino acids. Although a single set of kinetic parameters was obtained by kinetic experiments, other evidence indicates that transport systems for both the aromatic and the branched-chain amino acids serve for azaleucine. Azaleucine uptake in strain EO317, with a mutation leading to derepression and constitutive expression of branched-chain amino acid (LIV) transport and binding proteins, was not repressed by growth with leucine as it was in parental strain EO300. Lesions in the aromatic amino acid transport system, aroP, also led to changes in the regulation of azaleucine uptake activity when cells were grown on phenylalanine. Experiments on the specificity of azaleucine uptake and exchange experiments with leucine and phenylalanine support the hypothesis that both LIV and aroP systems transport azaleucine. The ability of external azaleucine to exchange rapidly with intracellular leucine may be an important contributor to azaleucine toxicity. We conclude from these and other studies that at least four other process may affect azaleucine sensitivity: the level of branched-chain amino acid biosynthetic enzymes; the level of leucine, isoleucine, and valine transport systems; the level of the aromatic amino acid, aroP, uptake system; and, possibly, the ability of the cell to racemize D and L amino acids. The relative importance of these processes in azaleucine sensitivity under various conditions is not known precisely.     

Absorption of neutral amino acids by the gut ofArenicola marina

Summary The absorption of neutral amino acids byArenicola marina was studied using anin vitro preparation of the alimentary canal. Regional variation in absorption was observed, with the intestine being the region of greatest uptake. The L enantiomorphs of the neutral amino acids alanine and leucine were shown to be actively absorbed by the intestine as was the D enantiomorph of alanine. A saturable component was demonstrated in the absorption of L-alanine and this was shared by L-methionine, which was found to competitively inhibit alanine uptake. Inhibition of L-alanine uptake also occurred in the presence of other neutral, basic and acidic amino acids. The greatest inhibition was found with the L stereoisomers of methionine, leucine, valine, histidine and phenylalanine, whilst proline, lysine and aspartic acid decreased uptake to a smaller extent.     

PERIODIC CHANGES IN RATE OF AMINO ACID UPTAKE DURING YEAST CELL CYCLE

Uptake of amino acids is a complex process but in cells growing with ammonia as sole nitrogen source the initial uptake rate of amino acids is a measure of the transport capacity of the uptake system (permease). In synchronous cultures of Saccharomyces cerevisiae amino acids were transported at all stages of the cell cycle. However, for any one amino acid the initial uptake rate was constant for most of the cycle and doubled during a discrete part of the cycle. Thus, for a variety of amino acids the functioning amino acid transport capacity of the membrane doubles once per cycle at a characteristic stage of the cycle. Arginine, valine, and phenylalanine exhibit periodic doubling of uptake rate at different stages of the cell cycle indicating that the transport of these amino acids is mediated by three different systems. Serine, phenylalanine, and leucine exhibit periodic doubling of the uptake rate at the same stage of the cycle. However, it is unlikely that serine and phenylalanine share the same transport system since the uptake of one is not inhibited by the other amino acid. This phenomenon is analogous to the periodic synthesis of soluble enzymes observed in S. cerevisiae.