The Transport of L-alanine by the Hamster Kidney Cell Line BHKWC13 D. M. SCOTT ' AND J. A. PATEMAN Department of Genetics, Institute of Genetics, university of Glasgow, Church Street, Glasgow GI 1 5JS, United Kingdom
ABSTRACT The uptake of L-alanine into BHK21-Cl3 cells i n culture has been studied. This amino acid appears to be transported essentially via a relatively low affinity, high capacity, sodium ion dependent transport system. Inhibition studies using other amino acids or their analogues provided information about the specificity of this system. This alanine transport system was shown to exhibit a broad substrate specificity and appeared to be capable of transporting most naturally occurring neutral a-amino acids. Kinetic studies of the inhibition of L-alanine uptake also indicated the presence of a second neutral amino acid transport system capable of transporting this amino acid. However, it is unlikely that this second uptake system contributes greatly to L-alanine uptake. Inhibition of the uptake of L-leucine indicated that this transport system has a similar specificity to the '%''-system initially described for Ehrlich ascites carcinoma cells. Amino acids constitute one of the largest and most diverse groups of compounds commonly concentrated by active transport, and their uptake has been studied in a wide variety of microbial and mammalian systems. Considerable progress has been made in the characterisation of the various amino acid transport systems in bacteria and fungi, but the number and specificity of similar transport systems in mammalian cells is less clear. The naturally occurring neutral L-a-amino acids appear to be transported by one or more systems of broad specificity both in mammalian tissues (Neame, '68; Heinz, '72; Slayman, '73) and in a large number of microorganisms (Oxender, '72; Slayman, '73). These amino acids also appear to be transported in microorganisms (Wargel e t al., '70; Brown, '70) and possibly in certain mammalian tissues (De Vries et al., '57; Hooft e t al., '68) via uptake systems specific for single amino acids or groups of structurally related amino acids. With the exception of the detailed studies by Christensen and coworkers with Ehrlich ascites carcinoma cells (Oxender and Christensen, '63; Christensen e t al., '65, '67; Inui a n d Christensen, '671, relatively few studies J. CELL. PHYSIOL. (1978)95: 57-64.
have been carried out to determine the characteristics of the active transport of neutral amino acids by either isolated mammalian cells or cells in culture. These latter authors demonstrated that certain of these amino acids may be transported by as many as three neutral amino acid uptake systems of completely or partially overlapping specificities (Oxender and Christensen, '63; Christensen e t al., '65, '67; Inui and Christensen, '67). The studies reported in this paper describe the transport characteristics of certain neutral amino acids in monolayer cultures of the baby hamster kidney cell line BHK21-Cl3.Lalanine, which has been previously shown to be transported via the three major neutral amino acid transport systems in Ehrlich ascites carcinoma cells (Christensen, '69) was consequently examined in most detail. This amino acid was demonstrated to be preferentially transported into BHK2l -C13 cells via a relatively low affinity, high capacity active transport system of broad specificity. HowReceived May 13, '77.Accepted Nov. 10, '77. ' Present address: Department of Medical Biochemistry, University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, United Kingdom.
57
58
D . M . SCOTT AND J. A. PATEMAN
ever, L-alanine also appeared to be capable of transport via a second “leucine preferring” uptake system. MATERIALS AND METHODS
Cell culture techniques Baby syrian hamster kidney cell line BHK21-C13 cells (Macpherson and Stoker, ’62; Macpherson, ‘63) were routinely grown in Glasgow modified Eagle’s minimum essential medium, GMEM (Macpherson and Stoker, ’62) supplemented with 10%(v/v) foetal calf serum (Gibco-Biocult Ltd., Paisley, Scotland). Cells were maintained at 37°C in an atmosphere of 5%CO, and 95%air. Prior to amino acid transport assay cells were harvested during exponential growth, inoculated into 60 mm petri dishes (Gibco-Biocult) a t a density of 5.3 x lo3 cells/cm’ and incubated as above for 48 hours. Uptake assay procedure Amino acid uptake was examined using a modification of the method described by Hatanaka et al. (‘69)for sugar transport studies. Following growth of the cells in petri dishes, the growth medium was removed by aspiration and the cells washed twice with warm (37°C) “uptake medium” of the following composition: Na C1 (114.8 mM), K Cl (5.4 mM), Mg SO, (0.8 mM), Ca C1, (1.8 mM), Na H,PO, (0.9 mM), Fe (N03)3(0.003mM), Na2 HPO, (0.9 mM), glucose (25 mM) and Hepes 2-(N-2- hydroxyethylpiperazin-N-y1)-ethanesulphonic acid (20 mM) buffer, pH 7.4. The “uptake medium” used for washing was removed and uptake medium (1ml), containing radioactively labelled amino acid (0.5-1 pCi/ ml) together with added cold carrier amino acid adjusted to the required initial concentration. The cultures were then incubated at 37°C for the required time interval, after which the “uptake medium” was removed by aspiration and the petri dish washed rapidly four times with warm (37°C) “uptake medium.” Accumulated radioactivity and the protein content of the cells were determined, following air-drying, utilising the Oyama and Eagle (’56) modification of the Lowry method (Lowry e t al., ’51)for protein estimation. Cells were treated with Folin’s solution (1ml), incubated for one hour a t 4”C, after which four aliquots (0.1 ml) were taken for protein determination. The radioactivity of four further aliquots were assayed in NE250 scintillation fluid (Nuclear Enterprises Ltd., NEN Chemi-
cal GMBH, Siemsstra e l , West Germany) using a Beckman L.511 liquid scintillation Spectrometer. Km and Vmax values for uptake of a n amino acid were determined by the method of weighted least squares regression as described by Wilkinson (‘61).Ki values for the inhibition of amino acid uptake were calculated from Kp values. These were determined from the transport of radioactive amino acid in the presence of a single concentration of inhibiting amino acid.
Extraction and ionophoresis of exogenous radioactive amino acids BHK21-Cl3 cells were grown in 60-mm petri dishes as described in Uptake assay procedure, and counted after trypsinisation of cells from duplicate petri dishes. Cells were labelled for five minutes at 37°C in “uptake medium” containing L- 12,3-3H1 alanine in the presence or absence of sodium azide (5 mM) and sodium cyanide (2 mM). The “uptake medium” was removed, the cells washed with warm non-radioactive “uptake medium” and then treated with cold trichloracetic acid (10% w/v). The cells were removed by scraping, and cellular material subsequently sedimented by centrifugation at 10,OOOg for 14 minutes. The free amino acids in the supernatant were then removed by ether extraction. L-alanine was separated from other amino acids in this extract by ionophoresis for three hours at 200 v in tank buffer, pH 1.9 (58 ml glacial acetic acid, 26 ml25%,v/v, formic acid, diluted to 21). After ionophoresis, strips (1 cm) were added to NE250 scintillation fluid for radioactivity determination. Determination of cell volume The mean cell volume of BHK21-Cl3 cells was determined by the method of Magath and Berkson (’60) using a model D Coulter Counter. The Coulter Counter was fitted with a 100 p aperture and calibrated using Ragweed pollen spores of known mean diameter. RESULTS
General characteristics of L-alanine uptake The incorporation of L-alanine into BHK21C13 cells was found to be linear over the first four minutes of assay (fig. 1 shows a typical plot of uptake over this period), for all concentrations of the amino acid examined. This increase in intracellular radioactivity was shown to be essentially due to the accumula-
59
L-ALANINE UPTAKE BY BHK21-CI3CELLS TABLE 1
Effect of inhibitors on L-alanine uptake in BHKZl-Cl3 cells Rate of L-alanine uptake (pmollug prnteinlmin)
Inhibitor
None Cycloheximide p-chloro-mercuribenzoate N-ethyl-maleimide 0u abain Sodium Cyanide Sodium azide
t
P
0.91 4.46 5.61 6.69 7.94 8.60
< 0.05
11.02 1.0 (1mM)
(100 pm) (100 pM) (100 pM) (2 mMf (5mM)
9.620.8 4.120.8 3.72 0.05 2.520.4 1.420.3 0.72 0.2
< 0.001 < 0.001
< 0.001 < 0.001 < 0.001
Uptake of i3HIL-alanine(200@MI was examined in the presence of the above inhibitors, following preincubation of cells in their presence for ten minutes. Rates of uptake were determined from 4-minute incubations. Values are means of six samples plus SEM. t values were calculated from comparison with control values, obtained prior to administration of inhibitor.
ber and the mean cell volume). No accumulation was observed in medium containing the inhibitors of mitochondria1 electron transfer, sodium azide (5 mM) and sodium cyanide (2 mM). Ouabain, an effective inhibitor of the magnesium-dependent sodium; potassium ATPase, and the sulphydryl group inhibitor pchloromercuribenzoate and N-ethylmaleimide were also demonstrated to be effective inhibitors of L-alanine uptake (table 1). In order to examine the characteristics of L-alanine uptake under various physiological conditions the effect of temperature, pH and sodium ion concentration on the transport of this amino acid were studied. L-alanine uprnin take was increased approximately 75-fold over the temperature range 1°C to an apparent Fig. 1 Initial rate of L-alanine and L-leucine uptake into BHK21-Cl3 cells. Cells were incubated at 37°C for in- optimum of 39°C (fig. 21, and exhibited a Ql0 tervals of up to four minutes in uptake medium containing value of 2.57 between 25°C and 37°C. The rate PHI-L-alanine(lOpM), ( 0 - 0 ) ;or PHI-L-leucine(10pM); of L-alanine uptake also varied with the pH of (0-0). the “uptake medium” (fig. 3). L-alanine uptake was seen to increase rapidly with increastion of radioactive L-alanine, as opposed to the ing pH between pH 5 and the apparent opmetabolism of this compound or its incorpora- timum a t approximately pH 7.6. The fact tion into protein. Extraction of the radioac- that L-alanine bears no net charge at these tively labelled cells with 10% (w/v) trichlor- pH’s would indicate that the observed pH acetic acid and its subsequent ionophoresis, dependence is a characteristic of the transport demonstrated that after four minutes incu- system and not due to changes in the charge of bation in medium containing L-L3H1 alanine the amino acid. Increased replacement of the (10 pM), 98%of the total radioactivity within sodium ion component of the medium sodium the cells remained in the trichloracetic acid chloride by choline chloride or D-mannitol soluble fraction and greater than 90% of this resulted in a marked reduction in the rate of was present as L-alanine. A comparison of the L-alanine uptake (fig. 4) thus indicating the intracellular and extracellular concentrations dependence of this transport system upon of L-13H1alanine indicated that L-alanine (10 sodium ions. pM) was concentrated within the cells apKinetics of L-alanine uptake proximately 50-fold within four minutes. (The The effect of concentration (S) on the initial intracellular concentration was determined from the radioactivity in the total cellular rate of uptake (v) is L-PHI alaine over the convolume, i.e., the product of the total cell num- centration range 10 pM to 4 mM was exam31
D.M. SCOTT AND J. A. PATEMAN
I 0
25
50
75 "a']
10
30
20
"C
Fig. 2 Effect of temperature on the uptake of L-alanine into BHK21-Cl3 cells. The uptake of PHI-L-alanine (200 pm) was examined a t various temperatures following a 10minute preincubation of the cells prior to assay. Rates of uptake were based on 4-minute incubations and each point represents the mean of six samples.
1
1 5
6
7
1 5
mEquivalentllitre
40
Temperature
4
100
8
9
PH
Fig. 3 Effect of pH on L-alanine uptake into BHK21C13 cells. The uptake of PHI-L-alanine (200 pM)was examined a t various pH's between 4.3 and 8.3. Initial rates of uptake were based on 4-minute incubations and each point represents the mean of six samples.
ined. The S/v versus S plot of this data was linear (fig. 5) indicating that the transport of L-alanine appeared to follow Michaelis-Menten kinetics and that this amino acid was apparently transported via a single system over this concentration range. Statistically determined estimates of the apparent Km and Vmax values gave values of 0.81 & 0.16 mM and 57.7 .t 4.1 pmol/pg protein/min respectively.
Fig. 4 Effect of sodium ion concentration on L-alanine uptake into BHK21-Cl3 cells. The uptake of PHI-L-alanine (200 pM) from uptake medium containing various sodium ion concentrations, was examined for 4-minute incubations. The sodium chloride component of the uptake medium was replaced by equiosmolar quantities of D-mannitol or choline -0)represents uptake in the presence of Dchloride. (0 mannitol and ( 0 - 0 ) in the presence of choline chloride. Each point represents the mean of six samples.
Specificity of the L-atanine uptake system In order to determine the specificity of the L-alanine transport system of BHK21-Cl3 cells the ability of other amino acids (or amino acid analogues) to inhibit L-alanine uptake was investigated. L-PHI alanine (100 pM) was examined in the presence of a 25-fold molar excess of inhibiting amino acids or their analogues (table 2). With the exception of L-cysteine and L-cystine all naturally occurring neutral L-a-amino acids were effective inhibitors of L-alanine transport. The most effective inhibitors were the short chain aliphatic or hydroxyl substituted amino acids; and the branched chain aliphatic or aromatic amino acids were the least effective. Little inhibition was produced by the D-stereoisomers of L-alanine and serine, or the acidic and basic L-aamino acids. In order to determine if the observed inhibition was competitive or non-competitive, the inhibition of L-alanine transport by L-serine, L-methionine, L-glycine, L-leucine and L-phenylalanine was examined in more detail. S/v versus S plots of L-alanine uptake in the presence and absence of these amino acids were approximately parallel, indicating that the inhibition (at least for the examined amino acids) was competitive (fig. 5a shows typical inhibition of L-alanine uptake as represented by L-serine and Lleucine). As the neutral amino acids exhibited such
61
L-ALANINE UPTAKE BY BHK21-Cl3 CELLS TABLE 2
Inhibition of L-alanine and L-leucine uptake into BHK21-Cl3 cells by various amino acids and their analogues Inhibiting amino acid
1
2
3
4 mM
S Fig. 5 a S/v versus S plots of t-alanine uptake in the presence and absence of L-leucine or L-serine. Uptake of PHI-L-alanine into BHK21-Cl3 cells was examined in the absence ( 0 0 )and presence of 10 mM L-leucine (0-0 1 or 2 mM L-serine (A -A 1. Rates of uptake were basedon incubations of up to four minutes. b S/v versus S plots of L-leucine uptake in the presence and absence of L-alanine. Uptake of PHI-L-leucine into BHK21-Cl3cells was examined in the absence 10- 0 ) and presence of 10 mM L-alanine (0 -0). Rates of uptake were based on 1-minute incubations.
-
Percentage Percentage inhibition of inhibition of L-12, 3.3Hlalanine L-IG-3Hlleucine uptake uptake
L-alanine L-serine L-threonine L-valine L-leucine L-isoleucine L-phenylalanine Glycine L-tryptophan L-tyrosine L-histidine L-aspartate L-glutamate L-arginine L-lysine L-cysteine L-cystine L-methionine L-proline L-glutamine L-aspargine D-alanine D-serine N-methyl-DL-alanine N-acetyl. DL-alanine DL-aianine hydroxamate a-AIB a-methyl serine DL-alanine-methyl-ester I-amino-ethyl-ester Cycoleucine DL-Cycloserine
922 6 87-c 4
84-C4 31-C 3 35-c 5 342 3 25-C3 5624 26C 2 212 2 262 3 7 23 9 22 7 23 1122 1 2 23 7 22 6826 7424 6 8 25 602 3 2124 24rt 3 8 6 26
212 3 812 4 84-t 5 872 5
1424
472 5
5 5 26
722
1 5 23 8125 822 7 10C 3 421 9 22 1 4 22
Uptake of PHlalanine (100&MI or PHI leucine (100pM) was examined in the absence and presence of inhibiting amino acid (of concentration 2.5 mM for L-amino acids, or 5 mM for DL-amino acid analogues). Rates of uptake were determined for alanine and leucine for incubation periods of four minutes and one minute respectively. Values for the inhibition of L-alanine and L-leucineuptake represent the results obtained with six and five samples respectively, these values are expressed as the mean SEM.
*
serine) and ineffective (i.e., L-leucine and Lwide variation in their abilities to inhibit L- valine) inhibitors of L-13H1 alanine uptake alanine uptake, and more than one neutral (table 3).Initial rates of L-serine uptake were amino acid transport system has been re- based on 4-minute incubations, during which ported in Ehrlich ascites tumour cells (Oxen- transport was linear for the examined concender and Christensen, '63;Inui and Christen- tration range (10 p,M to 4 mM). Initial rates of sen, '67;Christensen et al., '65,'67)the possi- uptake for L-leucine and L-valine were based bility of additional neutral transport systems on 1-minute incubations, as for most of the w a s investigated. Evidence for more than one afore mentioned concentrations transport was neutral transport system is provided from dif- not linear for longer incubations (fig. 1shows ferences in the abilities of certain amino acids typical plot of uptake for L-leucine). The obto inhibit L-alanine and L-leucine uptake servations that: (a) the Km values for uptake (table Z), and a comparison of Km and Ki of L-alanine and L-serine and separately Lvalues for effective (i.e., L-alanine and L- leucine and L-valine, were similar to their Ki
62
D . M . SCOTT AND J. A. PATEMAN TABLE 3
Comparison between K m and Ki values for entry of amino acids into BHK2I 4 1 3 cells Ki values when acting as inhibitors o f Amino acid Km (mM)
L-alanine L-serine L-valine L-leucine
0.81t0.16 0.752 0.12 0.392 0.06 0.51t 0.10
L-alanine
L-serine
L-valine
L-leucine
-
0.992 0.28
-
10.632 1.58
0.68t0.18 1.922 0.47
values when acting as inhibitors to the transport of each other, and (b) the Km values of Lalanine and L-leucine were significantly different from the Ki values obtained for these amino acids when acting as inhibitors of the transport of the other amino acid, are consistent with the transport of L-alanine and Lserine via a single agency, whilst L-leucine and L-valine appear to be transported via a separate system. However, as L-alanine and Lleucine each appear t o be competitive inhibitors of the others transport (figs. 5a,b), it would appear that these amino acids show some affinity for the other major system. The limited affinity of L-alanine for the “leucine preferring transport system” was indicated by its limited inhibition of L-i3H1leucine uptake as compared to nonradioactive L-leucine, Lvaline or L-phenylalanine (table 2). These results would thus indicate the presence of at least two transport systems capable of transporting t h e neutral L-a-amino acids in BHK21-Cl3 cells. DISCUSSION
The data presented in this paper indicates that the uptake of L-alanine into BHK21-Cl3 cells is accomplished essentially via a relatively low affinity, high capacity, sodium ion dependent active transport system. This system is markedly stereospecific and shows greatest affinity for the short chain aliphatic or hydroxy-substituted amino acids, an observation similar to those reported for the “alanine preferring” (A) transport system of Ehrlich ascites carcinoma cells (Oxender and Christensen, ’63). “A-like’’ transport systems have also been reported or indicated in rat intestine (Munch, ’661, rat calvarium (Finerman and Rosenberg, ’66), kidney (Webber e t al., ’61; Webber, ’62), chick embryo heart cells (Gazzola et al., ‘72; Franchi-Gazzola et al., ’73) and the cells of the Chinese hamster line G3 (Hooper, personal communication). Further information about the structural requirements of the “alanine preferring”
-
-
0.61rt0.13
-
0.4220.15
transport system was provided by the use of structural analogues of substrate amino acids. These studies suggest a number of structural requirements for uptake via this system: (a) An unmodified a-carboxyl group is essential; (b) The a-hydrogen may be replaced by a methyl group; (c) Only certain modifications of the aamino group may be tolerated, e.g., N-acetylation almost completely abolished transport via this system, whereas N-methylation had little effect. Studies by Christensen and coworkers (Oxender and Christensen, ’63; Christensen e t al., ’65, ’67; Inui and Christensen, ’67) have demonstrated in Ehrlich ascites carcinoma cells two additional neutral amino acid transport systems: a sodium ion and pH independent “leucine-preferring” (L) transport system, and a sodium ion sensitive “ASC” system of preferred substrates alanine, serine and cysteine. Inhibition studies reported in this paper indicate a second neutral transport system in BHK21-C13 cells of specificity similar to the “L”-system of Ehrlich ascites carcinoma cells. L-methionine was found to be an effective inhibitor of both the “alanine” and “leucine preferring” transport systems of BHK21-Cl3 cells, an observation similar to those made for Ehrlich ascites carcinoma cells (Oxender and Christensen, ’63). Systems of similar specificity to the “leucine preferring” transport systems of BHK21-C13 and Ehrlich ascites carcinoma cells have also been reported in isolated epithelial cells (Reiser and Christensen, ’71), syrian hamster embryo cells in culture (Hare, ’671, human (Winter and Christensen, ’64) and pigeon erythrocytes (Eavensen and Christensen, ’67). Although transport systems similar to the ASC system in Ehrlich ascites carcinoma cells have been demonstrated in other cells, e.g., rabbit reticulocytes (Winter and Christensen, ’65)and pigeon erythrocytes (Eavensen and Christensen, ’67), it is unlikely that such a system is important in the uptake
L-ALANINE UPTAKE BY BHK21-Cl3 CELLS
of L-alanine in BHK21-Cl3 cells as this amino acid was very strongly inhibited by N-methylDL-alanine (which is not transported via this system). Christensen (’69)has suggested that where multiple neutral amino acid transport systems occur in mammalian cells they are probably not exclusive but have overlapping specificities. The observation in BHK21-C13 cells that L-leucine and L-alanine, (although mutually competitive inhibitors (exhibit Ki values very different to their Km values would be consistent with such a hypothesis. These studies clearly indicate the presence of at least two major neutral L-a-amino acid transport systems in monolayer cultures of baby hamster kidney, BHK21-C13 cells. These systems differ in their preferred substrates but appear to have overlapping substrate. LITERATURE CITED Brown, K. D. 1970 Formation of aromatic amino acid pools in Escherichia coli K-12. J. Bacteriol., 104: 177-188. Christensen, H. N. 1969 Some special kinetic problems of transport. Advan. Enzymol., 32: 1-19. Christensen, H. N., M. Liang and E. G. Archer 1967 A distinct Na+-requiringtransport system for alanine, serine, cysteine and similar amino acids. J. Biol. Chem., 242: 5237-5246. Christensen, H. N., D. L. Oxender, M. Liang and K. A. Vatz 1965 The use of n-methylation to direct the route of mediated transport of amino acids. J. Biol. Chem., 240: 3609-3616. Eavensen, E., and H. N. Christensen 1967 Transport systems for neutral amino acids in the pigeon erythrocyte. J. Biol. Chem., 242: 5386-5396. Finerman, G. A. M., and L. E. Rosenberg 1966 Amino acid transport in bone. Evidence for separate transport systems for neutral amino and imino acids. 3. Biol. Chem., 241: 1487-1493. Franchi-Gazzola, R., G. C. Gazzola, P. Ronchi, V. Saibene and G. G. Guidiotti 1973 Regulation of amino acid transport in chick embryo heart cells. 11. Adaptive control sites for the “A mediation”. Biochim. Biophys, Acta, 291: 245-556. Gazzola, G. C., R. Franchi, V. Saibene, P. Ronchi and G. G. Guidiotti 1972 Regulation of amino acid transport in chick embryo heart cells. I. Adaptive system of mediation for neutral amino acids. Biochim, Biophys. Acta, 266: 407-421. Hare, J. D. 1967 Location and characteristics of the phenylalanine transport mechanism in normal and Polyoma-transformed hamster cells. Cancer Res., 27: 2357-2363. Hatanaka, M., R. J. Huebner and R. V. Gilden 1969 Alterations in characteristics of sugar uptake by mouse cells transformed by murine sarcoma viruses. J. Nat. Cancer Inst., 43: 1091-1096. Heinz, E. 1972 Transport of amino acids by animal cells.
63
In: Metabolic Pathways. Vol. 6. L. E. Hokin, ed. Academic Press, New York, pp. 455-501. Hooft, C., D. Carton, J. Snoeck, J. Immermans, I. Antener, C. Van den Henda and W. Oyaert 1968 Further investigations in the Methione Malabsorption Syndrome. Helv. Paed, Acta, 23: 334-349. Inui, Y., and H. N. Christensen 1967 Discrimination of single transport systems. The Na’ sensitive transport of neutral amino acids in the Ehrlich cell. J. Gen. Physiol., 50: 203-224. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the Folin Phenol reagent. J. Biol. Chem., 193: 265-275. Macpherson, I. 1963 Characteristics of a hamster cell clone transformed by polyoma virus. J. Nat. Cancer Inst., 30: 795-806. Macpherson, I. A,, and M. Stoker 1962 Polyoma transformation of hamster cell clones-an investigation of genetic factors affecting cell competence. Virology, 16: 147-151. Magath, T. B., and J. Berkson 1960 Electronic blood-cell counting. 3. Exp. Med., 34: 203-213. Munck, B. G. 1966 Amino acid transport by the small intestine of the rat. The existence and specificity of the transport mechanism of imino acids and its relationship to the transport of glycine. Biochim. Biophys. Acta, 120: 97-103. Neame, K. D. 1968 A comparison of the transport systems for amino acids in brain, intestine, kidney and tumour. In: Progress in Brain Research. Vol. 29, Brain Barrier Systems. A. Lajtha and D. H. Ford, eds. Elsevier, Amsterdam, pp. 185-196. Oxender, D. L. 1972 Amino acid transport in microorganisms. In: Metabolic Pathways. Vol. 6. L. E. Hokin, ed. Academic Press, New York, pp. 133-185. Oxender, D. L., and H. N. Christensen 1963 Distinct mediating systems for the transport of neutral amino acids by the Ehrlich cell. J. Biol. Chem., 238: 3686-3699. Oyama, V. I., and H. Eagle 1956 Measurement of cell growth in tissue culture with a phenol reagent (FolinCiocalteau). Proc. SOC.Exp. Biol. and Med., 91: 305-307. Reiser, S., and P. A. Christiansen 1969 Intestinal transport of amino acids a s affected by sugars. Amer. J. Physiol., 216: 915-924. Slayman, C. W. 1973 The genetic control of membrane transport. In: Current Topics in Membranes and Transport. Vol. 4. F. Bronner and S. Kleinzeller, eds. Academic Press, New York. Wargel, R. J., C. A. Shadur and F. C. Neuhaus 1970 Mechanism of D-cycloserine action: transport systems for D-alanine, D-cycloserine, L-alanine and glycine. J. Bacteriol., 103: 778-788. Webber, W. A. 1962 Interactions of neutral and acidic amino acids in rental tubular transport. Amer. J. Physiol., 202: 577-583. Webber, W. A., 3. L. Brown and R. F. Pitts 1961 Interactions of amino acids in renal tubular transport. Amer. J. Physiol., 200: 380-386. Winter, C. G., and H. N. Christensen 1964 Migration of amino acid across the membrane of the human erythrocyte. J. Biol. Chem., 239: 872-878. Winter, C. G., and H. N. Christensen 1965 Contrasts in neutral amino acid transport by rabbit erythrocytes and reticulocytes. J. Biol. Chem., 240: 3594-3600.
ABSTRACT The uptake of L-alanine into BHK21-Cl3 cells i n culture has been studied. This amino acid appears to be transported essentially via a relatively low affinity, high capacity, sodium ion dependent transport system. Inhibition studies using other amino acids or their analogues provided information about the specificity of this system. This alanine transport system was shown to exhibit a broad substrate specificity and appeared to be capable of transporting most naturally occurring neutral a-amino acids. Kinetic studies of the inhibition of L-alanine uptake also indicated the presence of a second neutral amino acid transport system capable of transporting this amino acid. However, it is unlikely that this second uptake system contributes greatly to L-alanine uptake. Inhibition of the uptake of L-leucine indicated that this transport system has a similar specificity to the '%''-system initially described for Ehrlich ascites carcinoma cells. Amino acids constitute one of the largest and most diverse groups of compounds commonly concentrated by active transport, and their uptake has been studied in a wide variety of microbial and mammalian systems. Considerable progress has been made in the characterisation of the various amino acid transport systems in bacteria and fungi, but the number and specificity of similar transport systems in mammalian cells is less clear. The naturally occurring neutral L-a-amino acids appear to be transported by one or more systems of broad specificity both in mammalian tissues (Neame, '68; Heinz, '72; Slayman, '73) and in a large number of microorganisms (Oxender, '72; Slayman, '73). These amino acids also appear to be transported in microorganisms (Wargel e t al., '70; Brown, '70) and possibly in certain mammalian tissues (De Vries et al., '57; Hooft e t al., '68) via uptake systems specific for single amino acids or groups of structurally related amino acids. With the exception of the detailed studies by Christensen and coworkers with Ehrlich ascites carcinoma cells (Oxender and Christensen, '63; Christensen e t al., '65, '67; Inui a n d Christensen, '671, relatively few studies J. CELL. PHYSIOL. (1978)95: 57-64.
have been carried out to determine the characteristics of the active transport of neutral amino acids by either isolated mammalian cells or cells in culture. These latter authors demonstrated that certain of these amino acids may be transported by as many as three neutral amino acid uptake systems of completely or partially overlapping specificities (Oxender and Christensen, '63; Christensen e t al., '65, '67; Inui and Christensen, '67). The studies reported in this paper describe the transport characteristics of certain neutral amino acids in monolayer cultures of the baby hamster kidney cell line BHK21-Cl3.Lalanine, which has been previously shown to be transported via the three major neutral amino acid transport systems in Ehrlich ascites carcinoma cells (Christensen, '69) was consequently examined in most detail. This amino acid was demonstrated to be preferentially transported into BHK2l -C13 cells via a relatively low affinity, high capacity active transport system of broad specificity. HowReceived May 13, '77.Accepted Nov. 10, '77. ' Present address: Department of Medical Biochemistry, University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, United Kingdom.
57
58
D . M . SCOTT AND J. A. PATEMAN
ever, L-alanine also appeared to be capable of transport via a second “leucine preferring” uptake system. MATERIALS AND METHODS
Cell culture techniques Baby syrian hamster kidney cell line BHK21-C13 cells (Macpherson and Stoker, ’62; Macpherson, ‘63) were routinely grown in Glasgow modified Eagle’s minimum essential medium, GMEM (Macpherson and Stoker, ’62) supplemented with 10%(v/v) foetal calf serum (Gibco-Biocult Ltd., Paisley, Scotland). Cells were maintained at 37°C in an atmosphere of 5%CO, and 95%air. Prior to amino acid transport assay cells were harvested during exponential growth, inoculated into 60 mm petri dishes (Gibco-Biocult) a t a density of 5.3 x lo3 cells/cm’ and incubated as above for 48 hours. Uptake assay procedure Amino acid uptake was examined using a modification of the method described by Hatanaka et al. (‘69)for sugar transport studies. Following growth of the cells in petri dishes, the growth medium was removed by aspiration and the cells washed twice with warm (37°C) “uptake medium” of the following composition: Na C1 (114.8 mM), K Cl (5.4 mM), Mg SO, (0.8 mM), Ca C1, (1.8 mM), Na H,PO, (0.9 mM), Fe (N03)3(0.003mM), Na2 HPO, (0.9 mM), glucose (25 mM) and Hepes 2-(N-2- hydroxyethylpiperazin-N-y1)-ethanesulphonic acid (20 mM) buffer, pH 7.4. The “uptake medium” used for washing was removed and uptake medium (1ml), containing radioactively labelled amino acid (0.5-1 pCi/ ml) together with added cold carrier amino acid adjusted to the required initial concentration. The cultures were then incubated at 37°C for the required time interval, after which the “uptake medium” was removed by aspiration and the petri dish washed rapidly four times with warm (37°C) “uptake medium.” Accumulated radioactivity and the protein content of the cells were determined, following air-drying, utilising the Oyama and Eagle (’56) modification of the Lowry method (Lowry e t al., ’51)for protein estimation. Cells were treated with Folin’s solution (1ml), incubated for one hour a t 4”C, after which four aliquots (0.1 ml) were taken for protein determination. The radioactivity of four further aliquots were assayed in NE250 scintillation fluid (Nuclear Enterprises Ltd., NEN Chemi-
cal GMBH, Siemsstra e l , West Germany) using a Beckman L.511 liquid scintillation Spectrometer. Km and Vmax values for uptake of a n amino acid were determined by the method of weighted least squares regression as described by Wilkinson (‘61).Ki values for the inhibition of amino acid uptake were calculated from Kp values. These were determined from the transport of radioactive amino acid in the presence of a single concentration of inhibiting amino acid.
Extraction and ionophoresis of exogenous radioactive amino acids BHK21-Cl3 cells were grown in 60-mm petri dishes as described in Uptake assay procedure, and counted after trypsinisation of cells from duplicate petri dishes. Cells were labelled for five minutes at 37°C in “uptake medium” containing L- 12,3-3H1 alanine in the presence or absence of sodium azide (5 mM) and sodium cyanide (2 mM). The “uptake medium” was removed, the cells washed with warm non-radioactive “uptake medium” and then treated with cold trichloracetic acid (10% w/v). The cells were removed by scraping, and cellular material subsequently sedimented by centrifugation at 10,OOOg for 14 minutes. The free amino acids in the supernatant were then removed by ether extraction. L-alanine was separated from other amino acids in this extract by ionophoresis for three hours at 200 v in tank buffer, pH 1.9 (58 ml glacial acetic acid, 26 ml25%,v/v, formic acid, diluted to 21). After ionophoresis, strips (1 cm) were added to NE250 scintillation fluid for radioactivity determination. Determination of cell volume The mean cell volume of BHK21-Cl3 cells was determined by the method of Magath and Berkson (’60) using a model D Coulter Counter. The Coulter Counter was fitted with a 100 p aperture and calibrated using Ragweed pollen spores of known mean diameter. RESULTS
General characteristics of L-alanine uptake The incorporation of L-alanine into BHK21C13 cells was found to be linear over the first four minutes of assay (fig. 1 shows a typical plot of uptake over this period), for all concentrations of the amino acid examined. This increase in intracellular radioactivity was shown to be essentially due to the accumula-
59
L-ALANINE UPTAKE BY BHK21-CI3CELLS TABLE 1
Effect of inhibitors on L-alanine uptake in BHKZl-Cl3 cells Rate of L-alanine uptake (pmollug prnteinlmin)
Inhibitor
None Cycloheximide p-chloro-mercuribenzoate N-ethyl-maleimide 0u abain Sodium Cyanide Sodium azide
t
P
0.91 4.46 5.61 6.69 7.94 8.60
< 0.05
11.02 1.0 (1mM)
(100 pm) (100 pM) (100 pM) (2 mMf (5mM)
9.620.8 4.120.8 3.72 0.05 2.520.4 1.420.3 0.72 0.2
< 0.001 < 0.001
< 0.001 < 0.001 < 0.001
Uptake of i3HIL-alanine(200@MI was examined in the presence of the above inhibitors, following preincubation of cells in their presence for ten minutes. Rates of uptake were determined from 4-minute incubations. Values are means of six samples plus SEM. t values were calculated from comparison with control values, obtained prior to administration of inhibitor.
ber and the mean cell volume). No accumulation was observed in medium containing the inhibitors of mitochondria1 electron transfer, sodium azide (5 mM) and sodium cyanide (2 mM). Ouabain, an effective inhibitor of the magnesium-dependent sodium; potassium ATPase, and the sulphydryl group inhibitor pchloromercuribenzoate and N-ethylmaleimide were also demonstrated to be effective inhibitors of L-alanine uptake (table 1). In order to examine the characteristics of L-alanine uptake under various physiological conditions the effect of temperature, pH and sodium ion concentration on the transport of this amino acid were studied. L-alanine uprnin take was increased approximately 75-fold over the temperature range 1°C to an apparent Fig. 1 Initial rate of L-alanine and L-leucine uptake into BHK21-Cl3 cells. Cells were incubated at 37°C for in- optimum of 39°C (fig. 21, and exhibited a Ql0 tervals of up to four minutes in uptake medium containing value of 2.57 between 25°C and 37°C. The rate PHI-L-alanine(lOpM), ( 0 - 0 ) ;or PHI-L-leucine(10pM); of L-alanine uptake also varied with the pH of (0-0). the “uptake medium” (fig. 3). L-alanine uptake was seen to increase rapidly with increastion of radioactive L-alanine, as opposed to the ing pH between pH 5 and the apparent opmetabolism of this compound or its incorpora- timum a t approximately pH 7.6. The fact tion into protein. Extraction of the radioac- that L-alanine bears no net charge at these tively labelled cells with 10% (w/v) trichlor- pH’s would indicate that the observed pH acetic acid and its subsequent ionophoresis, dependence is a characteristic of the transport demonstrated that after four minutes incu- system and not due to changes in the charge of bation in medium containing L-L3H1 alanine the amino acid. Increased replacement of the (10 pM), 98%of the total radioactivity within sodium ion component of the medium sodium the cells remained in the trichloracetic acid chloride by choline chloride or D-mannitol soluble fraction and greater than 90% of this resulted in a marked reduction in the rate of was present as L-alanine. A comparison of the L-alanine uptake (fig. 4) thus indicating the intracellular and extracellular concentrations dependence of this transport system upon of L-13H1alanine indicated that L-alanine (10 sodium ions. pM) was concentrated within the cells apKinetics of L-alanine uptake proximately 50-fold within four minutes. (The The effect of concentration (S) on the initial intracellular concentration was determined from the radioactivity in the total cellular rate of uptake (v) is L-PHI alaine over the convolume, i.e., the product of the total cell num- centration range 10 pM to 4 mM was exam31
D.M. SCOTT AND J. A. PATEMAN
I 0
25
50
75 "a']
10
30
20
"C
Fig. 2 Effect of temperature on the uptake of L-alanine into BHK21-Cl3 cells. The uptake of PHI-L-alanine (200 pm) was examined a t various temperatures following a 10minute preincubation of the cells prior to assay. Rates of uptake were based on 4-minute incubations and each point represents the mean of six samples.
1
1 5
6
7
1 5
mEquivalentllitre
40
Temperature
4
100
8
9
PH
Fig. 3 Effect of pH on L-alanine uptake into BHK21C13 cells. The uptake of PHI-L-alanine (200 pM)was examined a t various pH's between 4.3 and 8.3. Initial rates of uptake were based on 4-minute incubations and each point represents the mean of six samples.
ined. The S/v versus S plot of this data was linear (fig. 5) indicating that the transport of L-alanine appeared to follow Michaelis-Menten kinetics and that this amino acid was apparently transported via a single system over this concentration range. Statistically determined estimates of the apparent Km and Vmax values gave values of 0.81 & 0.16 mM and 57.7 .t 4.1 pmol/pg protein/min respectively.
Fig. 4 Effect of sodium ion concentration on L-alanine uptake into BHK21-Cl3 cells. The uptake of PHI-L-alanine (200 pM) from uptake medium containing various sodium ion concentrations, was examined for 4-minute incubations. The sodium chloride component of the uptake medium was replaced by equiosmolar quantities of D-mannitol or choline -0)represents uptake in the presence of Dchloride. (0 mannitol and ( 0 - 0 ) in the presence of choline chloride. Each point represents the mean of six samples.
Specificity of the L-atanine uptake system In order to determine the specificity of the L-alanine transport system of BHK21-Cl3 cells the ability of other amino acids (or amino acid analogues) to inhibit L-alanine uptake was investigated. L-PHI alanine (100 pM) was examined in the presence of a 25-fold molar excess of inhibiting amino acids or their analogues (table 2). With the exception of L-cysteine and L-cystine all naturally occurring neutral L-a-amino acids were effective inhibitors of L-alanine transport. The most effective inhibitors were the short chain aliphatic or hydroxyl substituted amino acids; and the branched chain aliphatic or aromatic amino acids were the least effective. Little inhibition was produced by the D-stereoisomers of L-alanine and serine, or the acidic and basic L-aamino acids. In order to determine if the observed inhibition was competitive or non-competitive, the inhibition of L-alanine transport by L-serine, L-methionine, L-glycine, L-leucine and L-phenylalanine was examined in more detail. S/v versus S plots of L-alanine uptake in the presence and absence of these amino acids were approximately parallel, indicating that the inhibition (at least for the examined amino acids) was competitive (fig. 5a shows typical inhibition of L-alanine uptake as represented by L-serine and Lleucine). As the neutral amino acids exhibited such
61
L-ALANINE UPTAKE BY BHK21-Cl3 CELLS TABLE 2
Inhibition of L-alanine and L-leucine uptake into BHK21-Cl3 cells by various amino acids and their analogues Inhibiting amino acid
1
2
3
4 mM
S Fig. 5 a S/v versus S plots of t-alanine uptake in the presence and absence of L-leucine or L-serine. Uptake of PHI-L-alanine into BHK21-Cl3 cells was examined in the absence ( 0 0 )and presence of 10 mM L-leucine (0-0 1 or 2 mM L-serine (A -A 1. Rates of uptake were basedon incubations of up to four minutes. b S/v versus S plots of L-leucine uptake in the presence and absence of L-alanine. Uptake of PHI-L-leucine into BHK21-Cl3cells was examined in the absence 10- 0 ) and presence of 10 mM L-alanine (0 -0). Rates of uptake were based on 1-minute incubations.
-
Percentage Percentage inhibition of inhibition of L-12, 3.3Hlalanine L-IG-3Hlleucine uptake uptake
L-alanine L-serine L-threonine L-valine L-leucine L-isoleucine L-phenylalanine Glycine L-tryptophan L-tyrosine L-histidine L-aspartate L-glutamate L-arginine L-lysine L-cysteine L-cystine L-methionine L-proline L-glutamine L-aspargine D-alanine D-serine N-methyl-DL-alanine N-acetyl. DL-alanine DL-aianine hydroxamate a-AIB a-methyl serine DL-alanine-methyl-ester I-amino-ethyl-ester Cycoleucine DL-Cycloserine
922 6 87-c 4
84-C4 31-C 3 35-c 5 342 3 25-C3 5624 26C 2 212 2 262 3 7 23 9 22 7 23 1122 1 2 23 7 22 6826 7424 6 8 25 602 3 2124 24rt 3 8 6 26
212 3 812 4 84-t 5 872 5
1424
472 5
5 5 26
722
1 5 23 8125 822 7 10C 3 421 9 22 1 4 22
Uptake of PHlalanine (100&MI or PHI leucine (100pM) was examined in the absence and presence of inhibiting amino acid (of concentration 2.5 mM for L-amino acids, or 5 mM for DL-amino acid analogues). Rates of uptake were determined for alanine and leucine for incubation periods of four minutes and one minute respectively. Values for the inhibition of L-alanine and L-leucineuptake represent the results obtained with six and five samples respectively, these values are expressed as the mean SEM.
*
serine) and ineffective (i.e., L-leucine and Lwide variation in their abilities to inhibit L- valine) inhibitors of L-13H1 alanine uptake alanine uptake, and more than one neutral (table 3).Initial rates of L-serine uptake were amino acid transport system has been re- based on 4-minute incubations, during which ported in Ehrlich ascites tumour cells (Oxen- transport was linear for the examined concender and Christensen, '63;Inui and Christen- tration range (10 p,M to 4 mM). Initial rates of sen, '67;Christensen et al., '65,'67)the possi- uptake for L-leucine and L-valine were based bility of additional neutral transport systems on 1-minute incubations, as for most of the w a s investigated. Evidence for more than one afore mentioned concentrations transport was neutral transport system is provided from dif- not linear for longer incubations (fig. 1shows ferences in the abilities of certain amino acids typical plot of uptake for L-leucine). The obto inhibit L-alanine and L-leucine uptake servations that: (a) the Km values for uptake (table Z), and a comparison of Km and Ki of L-alanine and L-serine and separately Lvalues for effective (i.e., L-alanine and L- leucine and L-valine, were similar to their Ki
62
D . M . SCOTT AND J. A. PATEMAN TABLE 3
Comparison between K m and Ki values for entry of amino acids into BHK2I 4 1 3 cells Ki values when acting as inhibitors o f Amino acid Km (mM)
L-alanine L-serine L-valine L-leucine
0.81t0.16 0.752 0.12 0.392 0.06 0.51t 0.10
L-alanine
L-serine
L-valine
L-leucine
-
0.992 0.28
-
10.632 1.58
0.68t0.18 1.922 0.47
values when acting as inhibitors to the transport of each other, and (b) the Km values of Lalanine and L-leucine were significantly different from the Ki values obtained for these amino acids when acting as inhibitors of the transport of the other amino acid, are consistent with the transport of L-alanine and Lserine via a single agency, whilst L-leucine and L-valine appear to be transported via a separate system. However, as L-alanine and Lleucine each appear t o be competitive inhibitors of the others transport (figs. 5a,b), it would appear that these amino acids show some affinity for the other major system. The limited affinity of L-alanine for the “leucine preferring transport system” was indicated by its limited inhibition of L-i3H1leucine uptake as compared to nonradioactive L-leucine, Lvaline or L-phenylalanine (table 2). These results would thus indicate the presence of at least two transport systems capable of transporting t h e neutral L-a-amino acids in BHK21-Cl3 cells. DISCUSSION
The data presented in this paper indicates that the uptake of L-alanine into BHK21-Cl3 cells is accomplished essentially via a relatively low affinity, high capacity, sodium ion dependent active transport system. This system is markedly stereospecific and shows greatest affinity for the short chain aliphatic or hydroxy-substituted amino acids, an observation similar to those reported for the “alanine preferring” (A) transport system of Ehrlich ascites carcinoma cells (Oxender and Christensen, ’63). “A-like’’ transport systems have also been reported or indicated in rat intestine (Munch, ’661, rat calvarium (Finerman and Rosenberg, ’66), kidney (Webber e t al., ’61; Webber, ’62), chick embryo heart cells (Gazzola et al., ‘72; Franchi-Gazzola et al., ’73) and the cells of the Chinese hamster line G3 (Hooper, personal communication). Further information about the structural requirements of the “alanine preferring”
-
-
0.61rt0.13
-
0.4220.15
transport system was provided by the use of structural analogues of substrate amino acids. These studies suggest a number of structural requirements for uptake via this system: (a) An unmodified a-carboxyl group is essential; (b) The a-hydrogen may be replaced by a methyl group; (c) Only certain modifications of the aamino group may be tolerated, e.g., N-acetylation almost completely abolished transport via this system, whereas N-methylation had little effect. Studies by Christensen and coworkers (Oxender and Christensen, ’63; Christensen e t al., ’65, ’67; Inui and Christensen, ’67) have demonstrated in Ehrlich ascites carcinoma cells two additional neutral amino acid transport systems: a sodium ion and pH independent “leucine-preferring” (L) transport system, and a sodium ion sensitive “ASC” system of preferred substrates alanine, serine and cysteine. Inhibition studies reported in this paper indicate a second neutral transport system in BHK21-C13 cells of specificity similar to the “L”-system of Ehrlich ascites carcinoma cells. L-methionine was found to be an effective inhibitor of both the “alanine” and “leucine preferring” transport systems of BHK21-Cl3 cells, an observation similar to those made for Ehrlich ascites carcinoma cells (Oxender and Christensen, ’63). Systems of similar specificity to the “leucine preferring” transport systems of BHK21-C13 and Ehrlich ascites carcinoma cells have also been reported in isolated epithelial cells (Reiser and Christensen, ’71), syrian hamster embryo cells in culture (Hare, ’671, human (Winter and Christensen, ’64) and pigeon erythrocytes (Eavensen and Christensen, ’67). Although transport systems similar to the ASC system in Ehrlich ascites carcinoma cells have been demonstrated in other cells, e.g., rabbit reticulocytes (Winter and Christensen, ’65)and pigeon erythrocytes (Eavensen and Christensen, ’67), it is unlikely that such a system is important in the uptake
L-ALANINE UPTAKE BY BHK21-Cl3 CELLS
of L-alanine in BHK21-Cl3 cells as this amino acid was very strongly inhibited by N-methylDL-alanine (which is not transported via this system). Christensen (’69)has suggested that where multiple neutral amino acid transport systems occur in mammalian cells they are probably not exclusive but have overlapping specificities. The observation in BHK21-C13 cells that L-leucine and L-alanine, (although mutually competitive inhibitors (exhibit Ki values very different to their Km values would be consistent with such a hypothesis. These studies clearly indicate the presence of at least two major neutral L-a-amino acid transport systems in monolayer cultures of baby hamster kidney, BHK21-C13 cells. These systems differ in their preferred substrates but appear to have overlapping substrate. LITERATURE CITED Brown, K. D. 1970 Formation of aromatic amino acid pools in Escherichia coli K-12. J. Bacteriol., 104: 177-188. Christensen, H. N. 1969 Some special kinetic problems of transport. Advan. Enzymol., 32: 1-19. Christensen, H. N., M. Liang and E. G. Archer 1967 A distinct Na+-requiringtransport system for alanine, serine, cysteine and similar amino acids. J. Biol. Chem., 242: 5237-5246. Christensen, H. N., D. L. Oxender, M. Liang and K. A. Vatz 1965 The use of n-methylation to direct the route of mediated transport of amino acids. J. Biol. Chem., 240: 3609-3616. Eavensen, E., and H. N. Christensen 1967 Transport systems for neutral amino acids in the pigeon erythrocyte. J. Biol. Chem., 242: 5386-5396. Finerman, G. A. M., and L. E. Rosenberg 1966 Amino acid transport in bone. Evidence for separate transport systems for neutral amino and imino acids. 3. Biol. Chem., 241: 1487-1493. Franchi-Gazzola, R., G. C. Gazzola, P. Ronchi, V. Saibene and G. G. Guidiotti 1973 Regulation of amino acid transport in chick embryo heart cells. 11. Adaptive control sites for the “A mediation”. Biochim. Biophys, Acta, 291: 245-556. Gazzola, G. C., R. Franchi, V. Saibene, P. Ronchi and G. G. Guidiotti 1972 Regulation of amino acid transport in chick embryo heart cells. I. Adaptive system of mediation for neutral amino acids. Biochim, Biophys. Acta, 266: 407-421. Hare, J. D. 1967 Location and characteristics of the phenylalanine transport mechanism in normal and Polyoma-transformed hamster cells. Cancer Res., 27: 2357-2363. Hatanaka, M., R. J. Huebner and R. V. Gilden 1969 Alterations in characteristics of sugar uptake by mouse cells transformed by murine sarcoma viruses. J. Nat. Cancer Inst., 43: 1091-1096. Heinz, E. 1972 Transport of amino acids by animal cells.
63
In: Metabolic Pathways. Vol. 6. L. E. Hokin, ed. Academic Press, New York, pp. 455-501. Hooft, C., D. Carton, J. Snoeck, J. Immermans, I. Antener, C. Van den Henda and W. Oyaert 1968 Further investigations in the Methione Malabsorption Syndrome. Helv. Paed, Acta, 23: 334-349. Inui, Y., and H. N. Christensen 1967 Discrimination of single transport systems. The Na’ sensitive transport of neutral amino acids in the Ehrlich cell. J. Gen. Physiol., 50: 203-224. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the Folin Phenol reagent. J. Biol. Chem., 193: 265-275. Macpherson, I. 1963 Characteristics of a hamster cell clone transformed by polyoma virus. J. Nat. Cancer Inst., 30: 795-806. Macpherson, I. A,, and M. Stoker 1962 Polyoma transformation of hamster cell clones-an investigation of genetic factors affecting cell competence. Virology, 16: 147-151. Magath, T. B., and J. Berkson 1960 Electronic blood-cell counting. 3. Exp. Med., 34: 203-213. Munck, B. G. 1966 Amino acid transport by the small intestine of the rat. The existence and specificity of the transport mechanism of imino acids and its relationship to the transport of glycine. Biochim. Biophys. Acta, 120: 97-103. Neame, K. D. 1968 A comparison of the transport systems for amino acids in brain, intestine, kidney and tumour. In: Progress in Brain Research. Vol. 29, Brain Barrier Systems. A. Lajtha and D. H. Ford, eds. Elsevier, Amsterdam, pp. 185-196. Oxender, D. L. 1972 Amino acid transport in microorganisms. In: Metabolic Pathways. Vol. 6. L. E. Hokin, ed. Academic Press, New York, pp. 133-185. Oxender, D. L., and H. N. Christensen 1963 Distinct mediating systems for the transport of neutral amino acids by the Ehrlich cell. J. Biol. Chem., 238: 3686-3699. Oyama, V. I., and H. Eagle 1956 Measurement of cell growth in tissue culture with a phenol reagent (FolinCiocalteau). Proc. SOC.Exp. Biol. and Med., 91: 305-307. Reiser, S., and P. A. Christiansen 1969 Intestinal transport of amino acids a s affected by sugars. Amer. J. Physiol., 216: 915-924. Slayman, C. W. 1973 The genetic control of membrane transport. In: Current Topics in Membranes and Transport. Vol. 4. F. Bronner and S. Kleinzeller, eds. Academic Press, New York. Wargel, R. J., C. A. Shadur and F. C. Neuhaus 1970 Mechanism of D-cycloserine action: transport systems for D-alanine, D-cycloserine, L-alanine and glycine. J. Bacteriol., 103: 778-788. Webber, W. A. 1962 Interactions of neutral and acidic amino acids in rental tubular transport. Amer. J. Physiol., 202: 577-583. Webber, W. A., 3. L. Brown and R. F. Pitts 1961 Interactions of amino acids in renal tubular transport. Amer. J. Physiol., 200: 380-386. Winter, C. G., and H. N. Christensen 1964 Migration of amino acid across the membrane of the human erythrocyte. J. Biol. Chem., 239: 872-878. Winter, C. G., and H. N. Christensen 1965 Contrasts in neutral amino acid transport by rabbit erythrocytes and reticulocytes. J. Biol. Chem., 240: 3594-3600.
