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.     

Evidence for a direct role of tRNA in an amino acid transport system.

The transport of phenylalanine by the general aromatic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA. Neither periodate-treated tRNA nor phenylalanine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake. Among preparations of specific tRNAs, tRNAPhe and tRNATyr were effective in stimulating the uptake of phenylalanine and tyrosine, respectively, and tRNAGlu and tRNAVal gave no detectable stimulation of phenylalanine or tyrosine transport. The preparation of tRNATyr was 10 times as active as unfractionated tRNA and gave as much as 167% stimulation of tyrosine transport. Correspondingly, the preparation of tRNAPhe was at least 3.5 times as active as the unfractionated tRNA and 2.5 times as active as the preparation of tRNATyr in stimulation of phenylalanine transport. Preliminary results in fractionation of the active component of tRNA for stimulating phenylalanine uptake show that the major activity resides in minor isoacceptor(s) tRNAPhe rather than the major component tRNAPhe, and the slight activity of preparations of tRNATyr is probably due to a contamination of the active tRNAPhe. Other preliminary results indicate that this type of stimulation occurs with uptake of other amino acids and their tRNA.     

Regulation of amino acid transport in L6 muscle cells: I. Stimulation of transport system a by amino acid deprivation

The regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na⁺-dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α-amino isobutyric acid (AIB), and by α-methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid-deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6-fold stimulation after 6 hours. The basal and stimulated transport were equally Na⁺-dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9-fold in deprived cells. Amino acid-deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline-supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline-deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly⁺ or L exclusively were ineffective. The stimulation of the transport-rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid-depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation.     

Regulation of amino acid transport system L by amino acid availability in CHO-K1 cells. A special role for leucine

Starvation of CHO-K1 cells for leucine leads to a 3–4-fold increase in transport system L activity, without modification of transport through systems A and ASC. The concentration of leucine must be below 10 μM before the enhancement of transport can be clearly seen. To achieve low concentrations of leucine such as 10 μM, extensive dialysis of fetal calf serum was required. The enhancement of transport was completed after 12–24 h of starvation and was fully reversed within 1 h of re-feeding with leucine. Starvation for isoleucine, valine or phenylalanine also produced an increase in system L transport activity, but the effect was only one half of that seen following leucine starvation.     

Monoiodo-d-tyrosine,an artificial amino acid radiopharmaceutical for selective measurement of membrane amino acid transport in the pancreas

Using ¹¹C-labeled natural amino acids, the functional diagnosis of tissue metabolism has been actively studied. Our interest has been focused on developing a clinically available ¹²³I-labeled artificial amino acid with a single metabolic function. For this study, [¹²³I]3-iodo-d-tyrosine ([¹²³I]d-MIT) was selected. In vitro and in vivo studies using ¹²⁵I-labeled d-MIT indicated that it showed a high pancreatic accumulation, selective affinity for membrane active transport systems, and was stable against enzymatic deiodination. A canine scintigraphic study using ¹²³I-labeled d-MIT and kinetic analysis showed that it behaved as an “artificial amino acid” radiopharmaceutical with selective membrane amino acid transport affinity in the pancreas.     

Third system for neutral amino acid transport in a marine pseudomonad.

Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system.     

Extended production of citric acid using an exchange filtration technique

A filtration technique has been applied to deep culture citric acid fermentation in an attempt to prolong the active phase of citric acid production. The fermentation was extended to 30 days with a daily production rate of between 9 and 10g^–1d^–1. A yield of over 70% citric acid was achieved.     

Maintenance of glucagon-stimulated system A amino acid transport activity in rat liver plasma membrane vesicles

Plasma membrane vesicles prepared from intact rat liver or isolated hepatocytes retain transport activity by systems A, ASC, N, and Gly. Selective substrates for these systems showed a Na+-dependent overshoot indicative of energy-dependent transport, in this instance, driven by an artificially-imposed Na+ gradient. Greater than 85% of Na+-dependent 2-aminoisobutyric acid (AIB) uptake was blocked by an excess of 2-(methylamino)isobutyric acid (MeAIB) with an apparent Ki of 0.6 mM. Intact hepatocytes obtained from glucagon-treated rats exhibited a stimulation of system A activity and plasma membrane vesicles isolated from those same cells partially retained the elevated activity. Transport activity induced by substrate starvation of cultured hepatocytes was also evident in membrane vesicles prepared from those cells. The membrane-bound glucagon-stimulated system A activity decays rapidly during incubation of vesicles at 4 degrees C (t1/2 = 13 h), but not at -75 degrees C. Several different inhibitors of proteolysis were ineffective in blocking the decay of transport activity. Hepatic system N transport activity was also elevated in plasma membrane vesicles from glucagon-treated rats, whereas system ASC was essentially unchanged. The results indicate that both glucagon and adaptive regulation cause an induction of amino acid transport through a plasma membrane-associated protein.     

Modulation of the activity of amino acid transport system L by phorbol esters and calmodulin antagonists in a human placental choriocarcinoma cell line.

We investigated the regulation of the activity of amino acid transport system L in the JAR human placental choriocarcinoma cell line by agents which are known to modulate the activities of three different classes of protein kinases, A-kinase, C-kinase and CaM-kinase. The system L activity was measured by determining the uptake of leucine in these cells, grown as confluent monolayers. Leucine uptake in these cells was predominantly Na(+)-independent, was stimulated by lowering the extracellular pH and was inhibited by hydrophobic neutral amino acids. These characteristics demonstrate that uptake of leucine in this cell line occurs primarily via system L. Treatment of the cells with cholera toxin and forskolin, agents which are known to elevate intracellular cAMP levels, did not have any effect on the activity of system L. 4 beta-Phorbol 12-myristate 13-acetate, an activator of C-kinase, but not the inactive analogue 4 alpha-phorbol 12,13-didecanoate, caused a significant stimulation of system L. The involvement of C-kinase in the phorbol ester-induced stimulation was supported by the finding that staurosporine, an inhibitor of C-kinase, effectively blocked the stimulation. Calmodulin antagonists, calmidazolium, W-7 and CGS 9343 B stimulated system L activity markedly. The potency of these antagonists was in the following order: calmidazolium greater than CGS 9343 B greater than W-7. This stimulatory effect was specific for system L because systems A and ASC were not stimulated by these agents. The stimulation caused by these agents was primarily due to an increase in the maximal velocity, the apparent Km of the system being only minimally affected. It is concluded that the activity of amino acid transport system L in the JAR human placental choriocarcinoma cell line is stimulated by C-kinase, inhibited by CaM-kinase and unaffected by A-kinase.     

A mathematical model for membrane transport of amino acid and Na+ in vesicles

Summary A model with a carrier having sites for both amino acid and Na⁺ can account for AIB (-aminoisobutyric acid) transport kinetics observed in membrane vesicles from SV3T3 (simian virus 40-tranformed Balb/c3T3 cells) and 3T3 (the parent cell line). The main feature of this cotransport model is that Na⁺ binding to carrier decreases the effectiveK _m for AIB transport, Na⁺ transport kinetics observed in both vesicle systems can be described by passive (possibly facilitated) diffusion. The lag of Na⁺ transport across the membrane compared to that for AIB, coupled to the Na⁺-dependent decrease in theK _m for AIB, accounts for the overshoot in intravesicular AIB observed for SV3T3 in the presence of an initial Na⁺ gradient. Extra-vesicular Na⁺ maintains a derease in theK _m for AIB influx before intra-vesicular Na⁺ has accumulated to balance it with a comparable decrease in theK _m for AIB efflux. 3T3 vesicles display little overshoot, and this finding can be explained mostly by a lower carrier affinity for Na⁺.     

Regulation of amino acid transport in L6 myoblasts. II. Different chemical properties of transport after amino acid deprivation

The mechanism of stimulation of amino acid transport system A caused by amino acid deprivation in L6 cells was investigated. In cells loaded with alpha-aminoisobutyric acid (AIB), amino acid deprivation increased the rate of proline uptake only after the intracellular [AIB] dropped below 7 mM. Efflux of proline was not sensitive to the presence of proline in the outer medium (with or without external Na+), suggesting that efflux through system A (and possibly uptake) is not susceptible to transinhibition. Transport (stimulated uptake) into amino acid-deprived cells and that into amino acid-supplemented cells differed in several chemical properties: 1) In the former group, transport was higher at lower pH values than in the latter, and the optimum pH values were 7.5 and 7.8, respectively. 2) Unlike proline uptake in supplemented cells, uptake in deprived cells was inhibited by 50% with N-ethylmaleimide (1 mM) or by 50 microM p-chloromercuribenzoate (PCMBS). Inhibition by PCMBS was not due to collapse of the Na+ gradient. The mercurial inhibited only the deprivation-induced stimulation of transport, bringing the rate of proline uptake to the "basal" uptake level observed in amino acid-supplemented cells. Proline uptake was not stimulated by a second deprivation following treatment with PCMBS and a supplementation-deprivation cycle. However, in untreated cells, or by reversing mercaptide formation with dithiotreitol, the second deprivation stimulated transport. Deprivation at 4 degrees C did not elicit stimulation of proline uptake. Cycloheximide prevented the stimulation and decreased the rate of proline uptake in deprived cells more efficiently than in supplemented cells. Actinomycin D prevented stimulation when added at the onset of deprivation. The above data indicate that stimulation of transport by deprivation is protein synthesis-dependent and that the stimulated transport had chemical properties distinct from the "basal" transport in supplemented cells. The evidence presented is consistent with a model of activation of a finite pool of transporters upon deprivation, the chemical characteristics of which differ from those of the "basal" transport system.     

Red-cell amino acid transport. Evidence for the presence of system ASC in mature human red blood cells.

The properties of Na+-dependent L-alanine transport in human erythrocytes were investigated using K+ as the Na+ substitute. Initial rates of Na+-dependent L-alanine uptake (0.2 mM extracellular amino acid) for erythrocytes from 22 donors ranged from 40 to 180 mumol/litre of cells per h at 37 degrees C. Amino acid uptake over the concentration range 0.1-8 mM was consistent with a single saturable component of Na+-dependent L-alanine transport. Apparent Km and Vmax. values at 37 and 5 degrees C measured in erythrocytes from the same donor were 0.27 and 0.085 mM respectively, and 270 and 8.5 mumol/litre of cells per h respectively. The transporter responsible for this uptake was identified as system ASC on the basis of cross-inhibition studies with a series of 42 amino acids and amino acid analogues. Apparent Ki values for glycine, L-alpha-amino-n-butyrate, L-serine and L-leucine as inhibitors of Na+-dependent L-alanine uptake at 37 degrees C were 4.2, 0.12, 0.16 and 0.70 mM respectively. Reticulocytes from a patient with inherited pyruvate kinase deficiency were found to have a 10-fold elevated activity of Na+-dependent L-alanine uptake compared with erythrocytes from normal donors. Separation of erythrocytes according to cell density (cell age) established that even the oldest mature erythrocytes retained significant Na+-dependent L-alanine transport activity. Amino acid transport was, however, a more sensitive indicator of cell age than acetylcholinesterase activity. Erythrocytes were found to accumulate L-alanine against its concentration gradient (distribution ratio approx. 1.5 after 4 h incubation), an effect that was abolished in Na+-free media. Na+-dependent L-alanine uptake was shown to be associated with L-alanine-dependent Na+ influx, the measured coupling ratio being 1:1.     

Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20.