Uptake of Lysine by Wild-Type and S-(2-Aminoethyl)-L-cysteine-Resistant Suspension-Cultured Cells of Triticum aestivum

The kinetics of uptake of L-lysine in wheat (Triticum aestivumcv. Chinese Spring) were analyzed in wild-type cells and inAEC-1 variant cells that are resistant to S-(2-aminoethyl)-L-cysteine(AEC). Uptake of lysine by AEC-1 cells was considerably slowerthan that by the wild-type cells. In the presence of carbonylcyanidem-chlorophenylhydrazone, the rates of uptake by both types ofcell were reduced to a similar linear component. Fitting theuptake data to one linear (diffusional) component and one Michaelis-Menten(active) system showed that, as compared to wild-type cells,AEC-1 cells have a reduced V_max and an increased K_m with respectto the active component, byt they have a similar diffusionalcomponent. Inhibition experiments with various amino acids indicatedthat the active component represents a carrier specific forbasic amino acids, which was competitively inhibited by AEC.The AEC-1 cells also showed reduced uptake of several neutraland acidic amino acids, but the rate of uptake of 3-O-methylglucosewas somewhat higher than that by wild-type cells. (Received May 16, 1989; Accepted September 4, 1989)     

Nitrate Assimilation in Higher Plants with Special Reference to the Cocklebur (Xanthium pennsylvanicum Wallr.)

A soluble NADH-dependent nitrate reductase is described forthe shoot system of Xanthium. Young leaves and immature stemtissues contain high levels of the enzyme. They are relativelyrich in free amino acids and amides but store little free nitrate.The specific activity of the enzyme is lower in fully expandedleaves, although these leaves exhibit higher rates of fixationof carbon in photosynthesis than do younger leaves. Neithernitrate nor free amino acids accumulate in the mesophyll ofthe leaf. Older parts of the stem axis accumulate large amountsof soluble nitrogen, almost entirely as free nitrate. Reservesof nitrate in the shoot and root are rapidly depleted if nitrateis removed from the external medium. Nitrate reductase is apparently absent from roots of Xanthium.This finding is supported by analyses of bleeding sap from nitrate-fedplants which show that 95 per cent of the nitrogen exportedfrom roots is present as free nitrate. However, roots are capableof synthesizing and exporting large amounts of amino nitrogenif supplied with reduced nitrogen such as urea or ammonium. A scheme is presented summarizing the main features of the metabolismof nitrate in Xanthium and this is compared with the situationin nitrate-fed plants of the field pea (Pisum arvense L.), aspecies previously shown to be capable of reducing nitrate inits root system.     

Free amino acid levels and the regulation of nitrate uptake in maize cell suspension cultures

The ability of individual amino acids to regulate nitrate uptakeand induction was studied in a Zea mays embryo cell line grownin suspension culture. The maize cells exhibited a marked preferencefor absorbing amino acids over nitrate when both were presentin culture medium. The addition of an individual amino acid(2 mM glutamine, glycine, aspartic acid, or arginine) to theculture medium with 1 mM nitrate completely inhibited nitrateuptake and resulted in a cycle of low levels of nitrate influxfollowed by efflux to the growth medium. Glutamine was readilyabsorbed by the cells and was particularly effective in supportingoptimum cell growth in the absence of an inorganic nitrogensource as compared to the three other amino acids evaluated.However, neither glutamine nor any of the remaining 19 proteinaceousamino acids appeared to be solely responsible for regulationof nitrate uptake and induction. The ability of amino acidsto regulate nitrate uptake and assimilation appears to be morerelated to their overall levels in the cell rather than to anaccumulation of a specific amino acid. Key words: Amino acids, nitrate uptake, maize, regulation, cell suspension culture     

Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium

Metabolic flux analysis is a useful tool for unraveling relationships between metabolism and cell function. Material balancing can be used to provide estimates of major metabolic pathway fluxes, provided all significant metabolite uptake and production rates are measured. Potential sources of metabolizable material in many serum-free media formulations are low molecular weight digests of biological material such as yeast extracts and plant or animal tissue hydrolysates. These digests typically contain large amounts of peptides, which may be utilized as amino acids. This article demonstrates the need for accounting for amino acids liberated from peptides in order to accurately estimate pathway fluxes in Chinese hamster ovary cells grown in a complex (hydrolysate containing) medium. A simplified model of central carbon metabolism provides the framework for analyzing external metabolite measurements. Redundant measurements are included to ensure the consistency of data and assumed biochemistry by comparing redundant measurements with their predicted values from a minimum data set, and by expressing the degree of agreement using a statistical "consistency index." The consistency index tests whether redundancies are satisfied within expected experimental error. For chemostat steady states of CHO cultures grown in a hydrolysate-supplemented medium, consistent data were obtained only when amino acids liberated from peptides were taken into account.     

The Uptake and Utilization of Bacteria, Amino Acids and Nucleic Acid Components by the Rumen Ciliate Eudiplodinium maggii

Washed suspensions of the rumen ciliate protozoon Eudiplodinium maggii grown in vitro and incubated anaerobically engulfed all the bacteria tested except for Bacteroides ruminicola and Klebsiella aerogenes. There was considerable variation (160–9100 bacteria/h/protozoon at an external concentration of 10¹⁰ bacteria/ml) in the rate at which the bacteria were engulfed, but Eu. maggii showed some preference for bacteria of rumen origin. Some of the bacteria were digested with the release of soluble materials into the medium. Free amino acids were incorporated from an 0.1 mM solution at rates of 0.13 to 0.84 pmol/h/protozoon. Evidence is presented that Eu. maggii could obtain half the amino acids required for growth by the engulfment and digestion of bacteria and half by the uptake of free amino acids. Eudiplodinium maggii incorporated uridine 5' monophosphate and also hydrolysed this to uridine and then to uracil which was reduced to dihydrouracil. These products all appeared in the medium. Ribose was incorporated by the protozoon and appeared as glucose in protozoal and bacterial polysaccharide; none was incorporated as such into protozoal nucleic acid.     

Utilization of dipeptides by Lactococcus lactis ssp. cremoris

Different strains of Lactococcus lactis ssp. cremoris hydrolyze peptides at different rates while the cell-free extracts of these strains all show the same or much higher rates of hydrolysis. These observations indicate that the uptake of peptides is the rate-limiting step in peptide hydrolysis. Utilization of leucyl-leucine by non-growing cells is competitively inhibited by the structurally related dipeptide alanyl-alanine. After hydrolysis of peptides, the amino acids are released into the medium and only a small fraction is accumulated and/or incorporated. This hydrolysis is independent of the synthesis of proteases indicating that the synthesis of proteases and peptidases are regulated differently. The specific growth rate of L. lactis ssp. cremoris E8 depends upon the amino acid source in the medium. No significant differences have been observed in the intracellular peptidase activities and the rates of peptide uptake between L. lactis ssp. cremoris E8 cells grown in different media, indicating that this growth rate is determined by the availability of amino acids in free amino acids or peptides.     

Mobilization of Endosperm Reserves and Uptake of Sucrose and Valine by the Cotyledons of Euphorbia lathyris L.

Upon germination, the endosperm triacylglycerols and proteinswere converted to sucrose and amino acids. During early postgerminativegrowth, the rate of sucrose and amino acid production exceededthe rate of uptake by the cotyledons. As a result, the levelsof total amino acid and sucrose in the endosperm increased;maximum levels were reached at 7 d and 10 d after imbibition(DAI), respectively. Intact seedlings were used to measure thedevelopment of valine, arginine, glutamic acid, and sucroseuptake rate throughout the course of endosperm depletion. Maximumamino acid uptake rates were measured at around 9 DAI, the highestuptake rate for sucrose was obtained at 12 DAI (just beforedepletion of the endosperm). The daily increase of sucrose andamino acid uptake could be manipulated, by replacing the endospermwith a pre-incubation solution during 1 d. The increase in sucroseuptake in vitro was equal to that measured with intact seedlingswhen the cotyledons were pre-incubated in 10 mol m^–3 sucrose.Higher sucrose concentrations reduced the increase of sucroseuptake; at 300 mol m^–3 sucrose (corresponding to the meanendosperm sucrose concentration) sucrose uptake after pre-incubationwas even lower than before. This reduction was largely counteractedwhen the pre-incubation solution was supplemented with minerals.The development of the valine uptake was hardly affected bysucrose, but was inhibited by several amino acids. Key words: Euphorbia lathyris seedling, sucrose uptake, amino acid uptake, reserve mobilization     

Effect of the Osmotic Environment on Assimilate Transport in Isolated Developing Embryos of Maize (Zea mays)

Embryos obtained from developing kernels of maize (Zea maysL.) were incubated in a bathing medium, to measure the effectof the osmotic environment on the balance between uptake andrelease of assimilates by the embryo. Net efflux of sucroseand amino acids from the embryo decreased with increasing mannitolconcentration in the bathing medium and net uptake of [¹⁴C]valine increased with increasing mannitol concentration. Therole of a high osmolality of the seed apoplast in seed developmentis discussed Zea mays, maize, embryo, seed development, assimilate transport, turgor-sensitive transport     

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.     

Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino acid transport.

The maximum specific growth rate of Streptococcus lactis and Streptococcus cremoris on synthetic medium containing glutamate but no glutamine decreases rapidly above pH 7. Growth of these organisms is extended to pH values in excess of 8 in the presence of glutamine. These results can be explained by the kinetic properties of glutamate and glutamine transport (B. Poolman, E. J. Smid, and W. N. Konings, J. Bacteriol. 169:2755-2761, 1987). At alkaline pH the rate of growth in the absence of glutamine is limited by the capacity to accumulate glutamate due to the decreased availability of glutamic acid, the transported species of the glutamate-glutamine transport system. Kinetic analysis of leucine and valine transport shows that the maximal rate of uptake of these amino acids by the branched-chain amino acid transport system is 10 times higher in S. lactis cells grown on synthetic medium containing amino acids than in cells grown in complex broth. For cells grown on synthetic medium, the maximal rate of transport exceeds by about 5 times the requirements at maximum specific growth rates for leucine, isoleucine, and valine (on the basis of the amino acid composition of the cell). The maximal rate of phenylalanine uptake by the aromatic amino acid transport system is in small excess of the requirement for this amino acid at maximum specific growth rates. Analysis of the internal amino acid pools of chemostat-grown cells indicates that passive influx of (some) aromatic amino acids may contribute to the net uptake at high dilution rates.     

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     

Changes in medium radioactivity and composition accompany high-affinity uptake of glutamate and aspartate by mouse brain slices

In measurements of high affinity transport in tissue slices, the incubation medium is often treated as an infinitely large pool. External substrate concentrations, even at the micromolar level, are assumed to be constant and metabolic interactions between tissue and medium are neglected. In the present report we describe experiments in which glutamic and aspartic acid uptake by mouse brain slices were studied using techniques that could test these assumptions. Cerebral hemispheres were cut into 0.1 mm sections and about 90 mg of tissue incubated in 10 ml of oxygenated medium. After 45 minutes of equilibration, radioactive substrates were added and the concentrations and specific activities of the amino acids and their metabolites in the medium were determined. During the first 10 min following substrate addition, rapid decreases in glutamic and aspartic acid concentrations in the medium were accompanied by large decreases in specific activity caused by the continuous release of these amino acids from the tissue. In addition, extensive conversion of both substrates to glutamine and the preferential accumulation of this metabolite, in the medium, was found. These results demonstrate that metabolism and release occur simultaneously with uptake during transport experiments in vitro and that these processes can take place in specific tissue compartments. It is therefore necessary to measure the tissue and medium concentration levels of amino acids along with their radioactivity in such experiments, since all three processes (transport, metabolism, and compartmentation) are interrelated in the clearance of amino acids from the incubation medium and probably from the extracellular spaces in vivo as well.     

UPTAKE SPECIFICITY FOR ORGANIC SUBSTRATES BY THE MARINE DIATOM MELOSIRA NUMMULOIDES1

Melosira nummuloides, clone Mel-3, shows a very high specificity with regard to its ability to take up organic substrates. Amino acids supplied in the medium at 1 X 10^-4 M are taken up at initial rates of the same order of magnitude as that of photoassirnilation of COj. However, sugars, sugar alcohols, or organic acids supplied at the same concentration are not taken up. The mechanism for uptake of amino acids appears to require energy, since tlie uptake of the amino acid analog α-aminoisobutyric acid is strongly inhibited by 2 f-dinitrophenul. The uptake mechanism does not appear to be inducible. The ability of M. numinuloides to utilize amino acids as a nitrogen source is quite restricted. Arginine, ghttamine, asparagine, proline, and glutamic acid were good nitrogen sources. Seventeen other amino acids, including α-aminoisobutyric acid, were unsatisfactory for growth, although they were rapidly taken up from the medium.     

Diel rhythmicity in amino acid uptake by Prochlorococcus

The marine cyanobacterium Prochlorococcus , the most abundant phototrophic organism on Earth, numerically dominates the phytoplankton in nitrogen (N)-depleted oceanic gyres. Alongside inorganic N sources such as nitrite and ammonium, natural populations of this genus also acquire organic N, specifically amino acids. Here, we investigated using isotopic tracer and flow cytometric cell sorting techniques whether amino acid uptake by Prochlorococcus is subject to a diel rhythmicity, and if so, whether this was linked to a specific cell cycle stage. We observed, in contrast to diurnally similar methionine uptake rates by Synechococcus cells, obvious diurnal rhythms in methionine uptake by Prochlorococcus cells in the tropical Atlantic. These rhythms were confirmed using reproducible cyclostat experiments with a light-synchronized axenic Prochlorococcus (PCC9511 strain) culture and ³⁵S-methionine and ³H-leucine tracers. Cells acquired the tracers at lower rates around dawn and higher rates around dusk despite >10⁴ times higher concentration of ammonium in the medium, presumably because amino acids can be directly incorporated into protein. Leucine uptake rates by cells in the S+G₂ cell cycle stage were consistently 2.2 times higher than those of cells at the G₁ stage. Furthermore, S+G₂ cells upregulated amino acid uptake 3.5 times from dawn to dusk to boost protein synthesis prior to cell division. Because Prochlorococcus populations can account from 13% at midday to 42% at dusk of total microbial uptake of methionine and probably of other amino acids in N-depleted oceanic waters, this genus exerts diurnally variable, strong competitive pressure on other bacterioplankton populations.     

Flower-Inducing Activity of Lysine in Lemna paucicostata 151 Cultured on Nitrogen-Deficient Medium

Lemna paucicostata 151, a weakly responsive short-day plant,flowers even under continuous illumination when cultured onnitrogen-free medium for more than 72 hours with subsequentculture on nitrogen-rich medium. During the nitrogen-free culture,the protease activity and protein content of the plant increasedand decreased, respectively. The plant contained a protein(s)that induced flowering of the plant when added to the medium.The level of this protein(s) also decreased during the nitrogen-freeculture. The total amount of free amino acids in plants culturedon nitrogen-free medium for 96 hours decreased to about 15%of that in plants at the start of nitrogen-free culture, butlevels of some amino acids increased. These amino acids wereexamined for their effects on flowering of plants cultured onnitrogen-deficient or nitrogen-free medium. Most of the aminoacids had no effect on flowering. However, profuse floweringwas induced when lysine was added to the medium. Lysine promotedthe flower-inductive process(es) rather than the developmentof flower buds. These results suggest that nitrogen deficiency-induced floweringof the plants is induced by lysine, which is generated froma specific protein(s) by proteolysis. (Received May 11, 1992; Accepted July 30, 1992)     

Energy-limitation of a sodium-independent amino acid transport in ovarian oocytes of the frog

The frog, Rana pipiens, hibernates through the winter with ovaries containing oocytes which, in size and appearance, are ready for ovulation and maturation. From November through April, the normal time of egg laying, ovulation and maturation can be induced by interrupting hibernation and administering gonadotropic hormones. In the studies reported here, it has been found that oocytes taken from hibernating animals in early winter take up amino acids from a saline medium at a relatively rapid rate. Respiratory inhibition produced by such agents as dinitrophenol (DNP) and anaerobiosis, does not completely stop uptake but slows it down markedly. In late winter, amino acid uptake tends to be slower in normal cells and when DNP or cyanide is added, a marked acceleration of the rate of uptake is observed. The uptake in poisoned cells is accumulative, producing internal concentrations higher than that of the medium. At this concentration of DNP, amino acid incorporation is almost completely stopped. Fluoride abolishes the DNP stimulation of amino acid uptake. Removal of sodium ion from the incubating medium has no effect on either uptake or incorporation. These data are interpreted to mean that a capacity for oxidative phosphorylation in oocytes diminishes during winter hibernation. In the spring either an anaerobic capacity comes into being or becomes capable of being switched on. During this time the rate of amino acid uptake, even in the downhill phase, is limited by energy availability and is not dependent on a sodium gradient.     

Amino acid uptake in Xenopus laevis oocytes.

The isolated oocytes from Xenopus laevis are able to take up radioactive amino acids from the exogenous medium. Most amino acids tested are taken up to reach concentrations higher than the extracellular medium. The initial uptake velocities vary with the external amino acid concentration in a Michaelis-Menten fashion. Aspartic acid requires concentrations an order of magnitude higher than the five other amino acids tested to reach half the maximal uptake velocity. The uptake mechanism seems to be specific for groups of analogous amino acids, as can be determined by competition studies. The amino acid groups for which there is some evidence of uptake specificity would be aromatic, aliphatic, acidic and basic. Amino acid pools of oocytes show that these cells can concentrate amino acids from Xenopus blood, as well as from artificial media.     

Effects of Nutrient Supply on Seed Growth of Field Bean (Vicia faba L.). 1. Effects of Different Sulphur and Methionine Regimes on Cotyledon Growth, Protein and Uncombined Amino Acids In vitro

The effects of differemt S and methionine regimes on growthof developing Vicia faba cotyledons in vitro were studied. Basalmedium (containing adequate S) supplemented with 05 mM methioninemarginally increased d. wt and uncombined amino acid accumulationbut adding 1–5 mM methionine inhibited both growth andprotein accumulation. Sulphur deficiency reduced both d. wtand protein accumulation but incresed accumulation of uncombinedamino acids. Adding 1 mM methionine to the S-deficient mediumrestored growth, normal protein and uncombined amino acid acnunulation.High sulphate medium (7.5 mM ) decreased d. wt, protein anduncombined amino acid accumulation. High sulphate medium or basal medium+methionine (05 mM) changedthe proportions of the seed proteins; legumin increased butvicilin decreased. Sulphur deficiency caused a relative increasein vicilin but a decrease in legumin. The different S and methionineregimes markedly changed the composition of the uncombined aminoacids, especially those derived from aspartic acid but not thecomposition of the protein fraction, except during S deficiency. The data presented indicates a flexibility in the storage proteincomposition of developing cotyledons grown in vitro, with theS and methionine status having a regulatory effect. Vicia faba L., field bean, cotyledon, growth, in vitro culture, uncombined amino acids, protein composition, legumin, vicilin, methionine, sulphur     

Die Bildung höherer Alkohole bei Aminosäuremangelmutanten von Saccharomyces cerevisiae

1. The influence of varying amounts of amino acids on the uptake of threonine, isoleucine, valine and leucine and their degradation to higher alcohols was investigated using a mutant strain of Saccharomyces cerevisiae, mating type a, genetic markers ade2, hom2, thr4, ilv2, leu1.
2. The cell mass is increased by increasing concentrations of threonine, isoleucine, valine and leucine, the latter two resulting in a higher dry weight. The amino acids are completely utilised at low concentrations. At higher contents up to 20% of the amino acids remain in the medium. The uptake of threonine, isoleucine, valine and leucine depends on the relative amounts of the concentrations of these amino acids in the medium. A greater amount of an amino acid is taken up if its concentration is comparatively higher than those of the other amino acids. There is a competition between the amino acids for the uptake into the cells.

Higher amounts of intracellular isoleucine and leucine are converted to 2-and 3-methylbutanol when compared with the degradation of valine and threonine to isobutanol and n-propanol-1, isoleucine and leucine up to 90%, valine up to 24% and threonine up to 20%. There is a competition between the four amino acids for their degradation to the corresponding higher alcohols. This behaviour confirms the earlier assumption of a degradation of the four amino acids by unspecific enzymes.     

Rapid microbial metabolism of non-protein amino acids in the sea

The non-protein amino acids, -alanine and -aminobutyric acid, frequently dominate the amino acid composition of deep-sea sediments. This accumulation is most likely due to the slower decomposition of non-protein amino acids by microorganisms or to the preferential adsorption of non-protein amino acids by clay minerals. We investigated relative rates of heterotrophic uptake of alanine, -ala, and -aba in sea water to see if there were different rates of microbial assimilation and respiration between these protein and non-protein amino acids. Heterotrophic uptake was rapid for all three amino acids with turnover times of hours in productive coastal waters and days in more oligotrophic open-ocean waters. Uptake of the non-protein amino acids was significantly slower than uptake of alanine, particularly in anoxic waters. However, the difference in uptake rates is probably not great enough to cause significant preferential accumulation of non-protein amino acids.