BIOCHIMICAET BIOPHYSICAACTA
741
RBA 75056
T R A N S P O R T OF AMINO ACIDS IN E H R L I C H ASCITES CELLS:
COMPETITIVE STIMULATION
JAMES A. SCHAFER
Department of Physiology, The University of Michigan, Ann Arbor, Mich.
AND JOHN A. JACQUEZ
Departments of Biostatistics and Physiology, The University of Michigan, Ann Arbor, Mich. ( U.S.A .)
(Received January 6th, I967)
(Revised manuscript received April I lth, 1967)
SUMMARY
I. The term 'competitive stimulation' refers to an experimental finding in
which the uptake of one amino acid is increased in the presence of another amino acid
when both are initially present extracellularly. The uptake of both amino acids of
such pairs was investigated with use of double-label techniques.
2. For all amino acid pairs examined which showed competitive stimulation,
the uptake of one amino acid was increased whereas that of the other was decreased.
Furthermore, the decrease in uptake of the latter was considerably greater than the
increase in uptake of the former.
3. In time studies it was found that the distribution ratio of the amino acid
whose uptake was stimulated was increased at all incubation times up to 3o rain.
The amino acid whose uptake was inhibited showed not only a markedly decreased
uptake but also a significantly altered curve of time dependence of uptake.
IlqTRODUCTION
The term 'competitive stimulation' has been applied to an experimental
finding with ascites tumor cells1, z in which the uptake of an amino acid is increased
in the presence of another amino acid when both are initially present extraceUularly.
CHRISTENSEN et al. 3 reported in 1952 that several L-dialnino acids increased the
steady-state distribution ratios of glycine, L-alanine and L-histidine. JACQUEZ4#
showed that several neutral L-amino acids as well as the amino acid analog azaserine,
increased the 2-min distribution ratio of L-tryptophan. He showed that although
stimulation of uptake of one amino acid occurred when both were present at approximately equimolar concentrations, inhibition of uptake of the stimulated
amino acid occurred when the concentration of the other amino acid was raised
sufficiently 5. In one experiment JACQUEZ4 measured the i-min uptakes of L-tryptoBiochim. Biophys. Acta, 135 (I967) 741-75 °
742
J.A. SCHAFER, J. A. JACQUEZ
phan and of azaserine, both present at the same initial extracellular concentration,
and noted that whereas the initial uptake of L-tryptophan was increased in the
presence of azaserine, the initial uptake of azaserine was decreased in the presence of
L-tryptophan, and that the decrease in uptake of azaserine was more than twice the
increase in uptake of L-tryptophan. As will be shown in this paper this has held true
for all pairs of amino acids showing competitive stimulation which we have examined.
In order to distinguish between the two amino acids in competitive stimulation and
to avoid the use of such long expressions as, 'the amino acid whose uptake was stimulated', and 'the amino acid whose uptake was inhibited', we will use the terms
'stimulated amino acid' and 'inhibited amino acid' in the remainder of this paper.
CHIRIGOS, FANNING AND GUROFF e and GUROFF, FANNING AND CHIRIGOS 2
reported that the neutral amino acid analog DL-p-fluorophenylalanine increased the
3o-min distribution ratios of L-tyrosine, L-p-aminophenylalanine, L-tryptophan, Lphenylalanine and L-lysine. GURO~F, FANNING AND CHIRIGOS2 also measured the
DL-p-fluorophenylalanine and L-tryptophan 3o-min distribution ratios in a competitive-stimulation experiment and found as did JACQUEZthat while the uptake of
L-tryptophan increased that of the DL-p-fluorophenylalanine decreased. However,
they reported that the increase in uptake of the L-tryptophan was approximately
equal to the decrease in uptake of the DL-p-fluorophenylalanine.
The stoichiometric relation between the changes in uptake of the two amino
acids in competitive stimulation is important for the discrimination of possible
carrier modelsS, ~. However, the information on relative changes in uptake of the
two amino acids rests on only two experiments4, 2 and they appear to give contradictory results. The present work was undertaken to provide measurements of
initial uptake rates as well as of the time course of uptake on a number of pairs of
amino acids which show competitive stimulation.
MATERIALS AND METHODS
In these experiments we used a hypotetraploid line of Ehrlich ascites carried
in this laboratory for approx. 300 generations b y weekly intraperitoneal inoculation
of o.I ml of ascites (diluted I :I or 2:1 with sterile saline) into female Swiss albino
mice. The collection and preparation of the ascites was conducted as described by
JACQUEZ AND SHERMAN1 with the exception that after each centrifugation the top
layer of the packed cells was removed by a suction pipette. After the final centrifugation the cells were resuspended to a cytocrit of o.18-o.32 in K R P solution (pH =
7.0) ~.
Design of experiments
All experiments involved two test amino acids each made to 4 mM in a separate
K R P solution. A fraction of the amino acid in one solution was labelled with 3H,
approx. 3 pC/ml, a fraction in the other solution with 14C, approx, o.15 pC/ml. In
18 experiments, referred to as 'competition studies' throughout this paper, 14 two* The solution referred to as KRP is a modified Krebs-Ringer phosphate buffer medium
of the followingcomposition: NaC1, 118.4 raM; KC1,4.73 mM; CaCIv o.84 raM; KHaPO4, 1.17 raM;
MgSO4•7H20, i. 17 mM; NaH~PO4•H20, 5-44 mM; Na2HPO4, i4. i5 raM. The pH of this buffer
was
consistently measured at 6.95-7.o4.
Biochim. Biophys. Acta, 135 (1967) 741-75°
COMPETITIVE
STIMULATION.
743
I
armed Heinicke tubes were set up as shown in Table I. All tubes were preincubated
for 3 mill at 37 °, then the solutions in the two arms were mixed b y tipping all tubes
simultaneously and incubated for 2 rnin at 37 °. Three time studies were also carried
out in which uptakes were measured after I, 5, IO, 2o and 3o min of incubation.
To terminate the incubations, the Heinicke tubes were plunged into ice-water
agitated to insure fast chilling. (Measurements with a thermocouple showed the temTABLE
I
PROTOCOL
Tube No.
FOR COMPETITION
A r m x of
Heinicke tube
Ascites
suspension
STUDIES
A r m * of Heinicke tube
K R P solution
(rot)
Amino acid z
(ml) (4 raM)
Amino acid 2
(ml) (4 raM)
(ml)
i
3.0
3.0
--
--
2-4
5, 6
3.0
3.0
1.5
--
1.5
3.0
---
7--9
IO, II
3 .0
3.O
1.5
--
---
1.5
3.O
I2--14
3.0
--
1. 5
1.5
perature of the solution inside the Heinicke tube reached 1 5 ° within I O - I I sec.) All
subsequent operations were conducted in a cold room at 2-4 °. The methods of
sample preparation and extraction were the same as those described in a previous
paper 1 with the following exception. The packed pellet of cells used for extraction
and analysis was obtained b y diluting i ml of cell suspension with 12 ml of cold
K R P solution and centrifuging in the cold. This modification was introduced to reduce
the correction for the amino acid retained in the extracellular space of the pellet.
Scintillation counting procedures
The analysis of sample activity was carried out with use of a Nuclear-Chicago
Model No. 725 liquid-scintillation system. For those samples which contained only
one isotope, the channels ratio procedure was used; the counting efficiencies were
54-60% for 14C and lO-14% for 3H. For samples which contained both I*C and 8H
the modification of the method of OKITA et al. 8 given in the Nuclear-Chicago instruction manual was followed. Our recoveries of 14C and 3H from samples containing both
always fell in the range 94-1o4°/o .
Calculations
The methods of calculation of the intracellular concentrations of the amino
acids have been described 4. The methods used in the present investigation differed
only in that the dilution of the extracellular concentrations in the pellets obtained
b y the modified sampling procedure had to be taken into account in the calculations.
All uptake data are given in terms of/tmoles of amino acid taken up per g dry weight
of cells.
Sources of materials
The unlabelled amino acids which were used were all grade-A amino acids
Biochim. Biophys. Acta,
i35
(1967)
741-75
o
744
J . A . SCHAFER, J. A. JACQUEZ
from California Biochemicals, except the DL-p-fluorophenylalanine which was obtained from K and K Laboratories in reagent grade.
The l*C-labelled amino acids used were: [2-14Clglycine , t-[i-14C]alanine, uniformly labelled L-[14Clleucine, and L-[ME-l*C]methionine, California Biochemicals;
L-[3-1*C]tryptophan, New England Nuclear; uniformly labelled L-[14Clphenylalanine ,
Nuclear-Chicago; I)L-p-fluoro[3-14Clphenylalanine, CEA France.
The tritiated amino acids used were: generally labelled L-[3H]methionine, generally labelled L-[3Hltryptophan, L-[ring-4-3H]phenylalanine, and generally labelled
L-[3H!phenylalanine, Nuclear-Chicago.
RESULTS
Competition studies
Table I I summarizes the results of 18 competition studies. Within each experiment, three 2-min uptakes were computed for both amino acids: the uptake of
the amino acid when alone at an initial extracellular concentration of I mM, the uptake of the amino acid from 2 mM extracellular concentration, and the uptake of the
given amino acid when at i mM with the second amino acid also present at i mM in
the extracellular fluid (in the table and often in the text this is referred to as the
I + I mM uptake). I t should be noted that all of the data reported in these experiments is in terms of averages: the uptakes of amino acids from i mM solutions represent the averages of three replicates, while the 2 mM uptakes are the averages of
two replicates. As would be expected, the variation between replicates was found to
be considerably less than that between experiments. The coefficient of variation
(standard deviation of mean divided b y the mean) averaged 2.99% for the means of
the triplicate determinations and 1.97% for the means of the duplicates. Also computed in Table I I is the sum of the uptakes of the two amino acids when both are
present at I mM, i.e. the sum of the I + I mM uptakes, this measures the 'total
amino acid' uptake in competition. This sum was compared to the sum of the uptakes of both amino acids when each was alone extracellularly at I mM. In all cases
this latter sum was greater than the sum of the competition I + I mM uptakes, indicating that even with competitive stimulation of one of the amino acids, the total
amino acid transported in competition is less than would be taken up if each amino
acid could be transported without interaction from the other amino acid. In addition,
the ratio of the uptake of each amino acid in the I + I mM competition situation to
the uptake when alone at an extracellular concentration of I mM was computed.
Any ratio greater than unity indicates that the particular amino acid is accumulated
to a greater extent in the presence of the second amino acid than in its absence. In
Table I I these ratios indicate t h a t competitive stimulation occurred in ten experiments; however, in no experiment was it found that the uptakes of both amino acids
of a pair were stimulated. In the remaining eight experiments, both amino acids
showed decreased uptake in the presence of the other amino acid.
The results of a single competitive-stimulation experiment, demonstrating the
salient facts seen in all such experiments, are illustrated in Fig. I. In the experiment
shown as well as in all the experiments of Table II, the total uptake from the i +
I mM mixture (last bar in Fig. i) is less than the uptake of the inhibited amino
acid from a 2 mM solution and is greater than the uptake of the stimulated amino
Biochim. Biophys. Acta, 135 (1967) 741-75 o
o
7
',D
,g
t~
OF TWO DIFFERENT
A M I N O ACIDS~ E A C H A T I
mM
EXTRACELLULAR
CONCENTRATION~
COMPARED
WITH
THE UPTAKES
OF EACH
L-[aH]Phe
L-~aH]Phe
L-[aH]Phe
15
23
28
L-[aH]Met
L-Jail]Met
L-Jail]Met
L-[SH]Trp
ii
i8
25
14
4.29
6.OI
L-[aH] Trp
i2
L-[aH] Phe
5.91
L-[SH]Trp
19
17
3.58
L-[SH]TrP
L-[aH]Trp
13
26
8.32
8.55
9.38
5. I I
2.68
3.87
3 -o8
3.59
2.60
2.81
L-[aH]Trp
L-[x4C]Trp
io
27
2.35
2.46
2.62
2.78
13.o7
12.76
13.79
7.69
7.26
8.98
9.5 °
4.67
3-69
4.79
3-91
4.43
3.56
3.27
5.19
4.19
4.68
4.46
A l o n e A lone
at
at
i mM 2 mM
7.51
6.62
7.20
3.49
4.79
5-49
6.59
2.8o
2.3 I
3 .°1
4.59
5.14
4.26
4.33
2.84
2.96
3.27
3.2o
z m M amino
acid I +
z m M amino
acid 2
Uptake o f amino acid z
L-[aH]Trp
L-[SH]Trp
L-[aH]Trp
L-[nH]Trp
Extracellular
amino acid •
3
4
5
9
Expt.
No.
9.55
11.34
lO.84
L-[14C]Ala
L-[x4C]Ala
DL-[14C]FPA
7.93
7.64
L-[t4C]Met
L-[xaC]Met
9.58
9.55
DL-[t4C]FPA
L-[x4C]Ala
9.48
L-[I~C]Ala
7 .66
L-Ei4C]Phe
lO.34
8.89
7.8o
DL-[x~C]FPA
DL-Ei4C]FPA
[x4C]Gly
8.65
8.22
6.84
5.96
8.46
6.26
A lone
at I m M
15-6o
17.58
15.36
15.96
11.17
11.44
14.75
15.72
14.21
1°.87
13.47
12.78
12.16
12.96
11.97
9.46
13.22
9.41
A lone
at 2 m M
9.36
8.82
8.12
9.61
6.o3
5.88
6.80
9.49
lO.14
4.73
4.96
4.o4
5.60
3.91
4.63
3-7 °
4.4 o
3.7 °
17.88
19.94
2o.22
14.69
12.22
13.65
15.46
13.o6
13.Ol
11.53
I 1.97
11.38
11.25
11.o4
I I.O8
9.o 4
8.42
9.19
16.86
15.44
15.32
13.1o
lO.82
11.37
13.39
12.3o
12.44
7-74
9-54
9.18
9.86
8-24
7"47
6.66
7.67
6.9o
(z + z)
B o th
present
at l m M
o.9o
o.77
0.77
o.68
1.12
o.91
1.12
0.78
0.86
0.78
1.49
1.43
1.64
1-54
1.21
1.2o
1.25
1.I5
Amino
acid I
o.98
o.78
o.75
I.OO
o.76
o.77
o.71
I.OO
0.98
0.62
o.56
o.52
o.65
o.48
0.68
0.62
o.52
0.59
Amino
acid 2
S u m o f uptakes o f
R a t i o o f uptakes
a m i n o acids x a n d 2 ( x + x ) m M / x m M
I mM amino
acid I +
Each
1 mM
alone
a m i n o acid 2 at x m M
Uptake of am i no acid 2
L-[x4C]Met
L-[aH]Met
L-[x4C]Leu
L-[I~C]Leu
L-[x'C]Leu
L-[x4C]Leu
ExtraceUular
a m i n o acid 2
Amino acid uptakes are in/~moles/g dr), wt. per 2 min incubation. All experiments were competition studies at 37 °. Abbreviation : FPA, DL-p-:fluorophenylalaine.
mM
UPTAKE
ALONE AT I AND 2
SIMULTANEOUS
TABLE II
call
z
0
v.¢
C
t"
t~
~,
t~
O
746
j . A . SCHAFER, J. A. JACQUEZ
Is.o F
,2.0
.~
L-METHIONINE
r
.0~-
- l ,ooy
SUM
EI~ 9.01=tJ ¢=" 8.0~<
I--
0.
D
z
7.c
6.C
5.C
4.C
~,.
3.C
N
2.C
L-TRYPTOPHAN
I.C
ImM 2raM I + l m M
EXTRACELLULAR
ImM 2raM l÷ImM
I + I mM
AMINO ACID CONCENTRATION
Fig. i. 2-min uptakes of L-tryptophan and L-methionine measured within the same competition
study. The bar labelled i mM denotes the uptake of the given amino acid from an extracellular
concentration of i mM alone, 2 mM denotes the uptake from a 2 mM concentration alone, while
i + i mM indicates uptake of the given amino acid from an extracellular solution containing
i mM of each of the amino acids. The bar on the far right gives the sum of the I + I mM uptakes of both amino acids.
acid from a 2 mM solution. The increase in u p t a k e of t h e s t i m u l a t e d a m i n o acid seen
in t h e I + I mM s i t u a t i o n is significantly less t h a n t h e decrease in u p t a k e of t h e
i n h i b i t e d a m i n o acid. This is in c o n t r a d i c t i o n (especially E x p t s . 13 a n d 26) to t h e
finding r e p o r t e d b y GUROFF, FANNING AND CttlRIGOS 2. Table I I I indicates t h a t t h i s
holds for all a m i n o acid pairs t e s t e d t h a t d e m o n s t r a t e c o m p e t i t i v e s t i m u l a t i o n : i.e.
t h a t t h e r a t i o of the decrease in u p t a k e of t h e i n h i b i t e d a m i n o acid to the increase in
u p t a k e of t h e s t i m u l a t e d a m i n o acid is always significantly g r e a t e r t h a n u n i t y .
A n o t h e r i m p o r t a n t o b s e r v a t i o n from Table I I I is t h a t in the e x p e r i m e n t s in w h i c h
L - t r y p t o p h a n u p t a k e was s t i m u l a t e d b y nL-p-fluorophenylalanine or L-methionine,
t h e increase in u p t a k e of L - t r y p t o p h a n p r o v i d e d b y I mM of t h e i n h i b i t e d a m i n o
acid (AUs,) was g r e a t e r t h a n the increase in u p t a k e o b t a i n e d b y increasing t h e conc e n t r a t i o n of L - t r y p t o p h a n b y I mM (AUsl). T h u s t h e a d d i t i o n of certain o t h e r a m i n o
acids can be more effective in increasing L - t r y p t o p h a n u p t a k e t h a n the a d d i t i o n of
more L - t r y p t o p h a n . C e r t a i n l y this o b s e r v a t i o n is c o n t r a r y to w h a t we would expect
from most of t h e simple carrier models which h a v e been used so far.
T i m e studies
Three e x p e r i m e n t s were c o n d u c t e d to d e t e r m i n e the s i m u l t a n e o u s t i m e course
of u p t a k e of two c o m p e t i n g a m i n o acids. A t each of five i n c u b a t i o n t i m e s t h e distrib u t i o n r a t i o of each a m i n o acid alone at I mM, a n d each a m i n o acid at I mM in
t h e presence of t h e o t h e r a m i n o acid also at I mM, were d e t e r m i n e d . I n t h e l a t t e r
case two replicates were used, in t h e former only one d e t e r m i n a t i o n was done because
of l i m i t a t i o n s in t h e n u m b e r of Heinicke t u b e s t h a t could be h a n d l e d a d e q u a t e l y in
a n y one e x p e r i m e n t .
Figs. 2 a n d 3 illustrate t h e t i m e course of t w o e x a m p l e s of c o m p e t i t i v e s t i m u lation. I n b o t h cases t h e s t i m u l a t e d a m i n o acid when alone is t a k e n u p more slowly
t h a n is the i n h i b i t e d a m i n o acid, which when alone shows a v e r y r a p i d initial u p t a k e
r a t e a n d p l a t e a u s w i t h i n a short time. E x a m i n i n g Figs. 2 a n d 3, it is seen t h a t in t h e
Biochim. Biophys. Acta, 135 (1967) 741-75 o
COMPETITIVE STIMULATION. I
747
TABLE I I I
COMPARISON
OF INCREASED
THE INHIBITED
UPTAKE
OF THE
STIMULATED
AMINO
ACID TO DECREASED
UPTAKE
OF
AMINO ACID
The data presented here is from the same experiments as that reported in Table I. Uptakes
are in/,moles/g dry wt. per 2 rain incubation. Abbreviation: FPA, nL-p-fluorophenylalanine.
Expt.
No.
Stimulated
amino acid
AUsl*
Aus***
Inhibited
amino acid
zluix"
AUl***
Izlut2[
IAu~,l
3
4
5
9
L-[aH]Trp
L-[nH]Trp
L-[SH]Trp
L-[SH]Trp
2.84
1.73
2.06
1.68
0.49
0.50
0.65
o.42
L-[x4C]Leu
L-[x4C]Leu
L-[14C]Leu
5.13
3.5 °
4.76
3.15
--2.21
--2.26
--4.06
--2.56
4.51
4.52
6.25
6.1o
IO
27
L-[SH]Trp
L-[liC]Trp
o.97
0.46
1.66
1.52
L-[x4C]Met
L-[3H]Met
3.51
4-74
--3.o5
--4.31
1.84
2.84
13
26
15
L-[SH]Trp
L-[SH]Trp
L-[SH]Met
o-83
o.84
3.59
1-51
1.55
0.68
DL-[x4C]FPA
DL-[x*C]FPA
DL-[14C]FPA
4.58
4.98
5.20
--3.93
--3.76
--2.75
2.61
2.42
4.05
L-[14C]Leu
" (uptake of amino acid from 2 mM alone) -- (uptake of amino acid from I mM alone).
** (uptake of amino acid from i mM with i mM of second amino acid present) -- (uptake
of amino acid from i mM alone).
I + I mM c o m p e t i t i o n s i t u a t i o n the i n i t i a l r a t e of u p t a k e of t h e i n h i b i t e d a m i n o acid
is m u c h decreased, a n d t h e i n t r a c e l l u l a r c o n c e n t r a t i o n p l a t e a u s a t a decreased level
a n d over a longer t i m e course. I n t h e c o m p e t i t i o n s i t u a t i o n t h e general shape of t h e
u p t a k e curve for t h e s t i m u l a t e d a m i n o acid does n o t seem t o be g r e a t l y a l t e r e d b u t
r a t h e r o n l y shifted u p w a r d from t h e I mM u p t a k e curve. The curves also illustrate
t h a t a t all i n c u b a t i o n t i m e s t h e decrease in u p t a k e of t h e i n h i b i t e d a m i n o acid is
g r e a t e r t h a n t h e increased u p t a k e of t h e s t i m u l a t e d a m i n o acid ill the I + I m M
s i t u a t i o n . This is especially true in t h e case shown in Fig. 3. F o r comparison, Fig. 4
shows a t i m e s t u d y in which c o m p e t i t i v e i n h i b i t i o n is observed. Note t h a t here, in
c o n t r a s t to t h e c o m p e t i t i v e - s t i m u l a t i o n t i m e studies, t h e u p t a k e curves of b o t h
a m i n o acids are m e r e l y depressed u n i f o r m l y in the presence of t h e second a m i n o acid.
The two curves for L-phenylalanine do n o t differ significantly a t 30 min.
DISCUSSION
A l t h o u g h e x p e r i m e n t a l o b s e r v a t i o n o f t h e p h e n o m e n o n of c o m p e t i t i v e stim u l a t i o n has been limited, several c h a r a c t e r i s t i c s a p p e a r t o be quite well established.
F o r several pairs of a m i n o acids showing c o m p e t i t i v e s t i m u l a t i o n t h e p e r cent s t i m u l a t i o n has been d e m o n s t r a t e d to be m a x i m a l when t h e s t i m u l a t i n g a m i n o acid is
a p p r o x i m a t e l y e q u i m o l a r w i t h respect to t h e s t i m u l a t e d a m i n o acidS, *. Secondly,
our results c o r r o b o r a t e those of GUROFF, FANNING AND CHIRIGOS 2 which i n d i c a t e
t h a t t h e i n h i b i t e d a m i n o acid is t a k e n u p m u c h more r a p i d l y a n d to higher s t e a d y s t a t e levels when alone e x t r a c e l l u l a r l y t h a n is t h e s t i m u l a t e d a m i n o acid u n d e r t h e
s a m e circumstances.
I n these e x p e r i m e n t s , we h a v e found, as h a v e others4, ~, t h a t w i t h those pairs
Biochim. Biophys. Acta, ~35 (1967) 741-75 °
748
J.A. SCHAFER, J. A. JACQUEZ
04f --
4.C
O
3.5
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8
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6 3.0
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r-
2.OllLI ¢: ,_,,-/-Ik'i
.......
8.......
,
°
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,
g=
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0.5
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0.51
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2
4 6
. . . . .
8 I0
;)0
30
TIME (MINUTES)
0
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20
30
TIME (MINUTES)
Fig. 2. T i m e d e p e n d e n c e of s i m u l t a n e o u s l y m e a s u r e d d i s t r i b u t i o n ratios for Dl.-p-fluorophen y l a l a n i n e (FPA) a n d L - t r y p t o p h a n for e a c h alone a t i m M a n d in t h e presence of t h e second
a m i n o acid also a t i m M . 0 - - 0 , DL-p-fluorophenylalanine w h e n alone a t i m M ; © - - - - - O ,
DL-p-fluorophenylalanine w h e n a t i m M w i t h i ~
L - t r y p t o p h a n ; × - - × , L : t r y p t o p h a n alone
a t I raM; [ Z - - - - - [ ~ ,
L - t r y p t o p h a n w h e n a t i m M w i t h DL-p-fluorophenylalanine also p r e s e n t
a t I mM. All 2 - m i n d i s t r i b u t i o n ratios are n o t f r o m t h e s a m e e x p e r i m e n t b u t are a v e r a g e d v a l u e s
f r o m c o m p e t i t i o n studies.
Fig. 3- T i m e d e p e n d e n c e of s i m u l t a n e o u s l y m e a s u r e d d i s t r i b u t i o n ratios for DL-p-fluorophenyla l a n i n e a n d L - p h e n y l a l a n i n e for e a c h alone a t I m M a n d in t h e presence of t h e second a m i n o
acid also a t z m M . O - - O , DL-p-fluorophenylalanine w h e n alone a t i m M ; O - - - - - G ,
DL-pf l u o r o p h e n y l a l a n i n e w h e n a t I m M w i t h L - p h e n y l a l a n i n e also p r e s e n t a t I m M ; × - - x , L-phenyla l a n i n e w h e n alone a t i raM; [ ] - - - - - - I N ,
L - p h e n y l a l a n i n e w h e n a t i m M w i t h DL-p-fluorop h e n y l a l a n i n e also p r e s e n t a t i m M . All 2-rain d i s t r i b u t i o n ratios are n o t f r o m t h e s a m e experim e n t b u t are a v e r a g e d v a l u e s f r o m c o m p e t i t i o n studies.
of amino acids exhibiting competitive stimulation there was a decrease in the uptake
of the inhibited amino acid concurrent with the increased uptake of the stimulated
amino acid. However, we have found that the decrease in uptake of the inhibited
amino acid is always considerably greater than the increase in uptake of the stimulated
amino acid whereas G u R o ~ , FANNING AND CHIRIGOS2 reported that the two
were approximately equal. The finding of GUROI~F, FANNING AND CHIRIGOS2 is based
on a 3o-min incubation, while the bulk of our data derives from 2-min initial uptakes;
nevertheless, our time studies show that the depression of nL-p-fluorophenylalanine
uptake far exceeds the increase in uptake of L-phenylalanine or L-tryptophan (Fig. 2)
out to 30 min of incubation. The time studies also suggest that the percentage
stimulation is greater at shorter incubation times.
GUROFF, FANNING AND CHIRIGOS~ have suggested that the stimulatory effect
is occurring extracellularly since they have found that there is a definite stimulation
of uptake within I rain of incubation, supposedly before either amino acid has an
appreciable intracellular concentration. Our studies also show significant stimulation
Biochim. Biophys. Acta, 135 (1967) 741-75 °
749
COMPETITIVE STIMULATION. I
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z
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ell
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1.0 7
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0 246810
5
~
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TIME(MINUTES)
30
Fig, 4. Time dependence of simultaneously measured distribution ratios for L-phenylalanine and
L-tryptophan for each alone at i mM and in the presence of the second amino acid also at i raM.
0---0, L-phenylalanine when alone at i mM; O - - - - - O ,
L-phenylalanine when at I mM with
I mM L-tryptophan also present; × - - × , L-tryptophan when alone at i raM; D - - - - - - U , Ltryptophan when at i mM with L-phenylalanine also present at i raM. All 2-min distribution
ratios are not from the same experiment but are averaged values from competition studies.
w i t h i n T min of i n c u b a t i o n ; however, b y I min t h e d i s t r i b u t i o n r a t i o for t h e inh i b i t e d a m i n o acid is far from negligible (2.O--3.T in t h e case of DL-p-fluorophenylalanine). GUROFF, FANNING AND CHIRIGOS2 h a v e also r e p o r t e d t h a t a short preincub a t i o n w i t h t h e i n h i b i t e d a m i n o acid gives a g r e a t e r s t i m u l a t o r y effect when t h e
second a m i n o acid is added. This would n o t only s u p p o r t t h e i r h y p o t h e s i s t h a t some
t i m e m u s t be a l l o t t e d for c o m b i n a t i o n of t h e i n h i b i t e d a m i n o acid a n d its e x t r a c e l l u l a r
site, b u t also t h e p o s s i b i l i t y t h a t it has to be a c c u m u l a t e d i n t r a c e l l u l a r l y to e x e r t its
effect. T h e y h a v e suggested an e x p l a n a t i o n of c o m p e t i t i v e s t i m u l a t i o n b a s e d on the
p o s t u l a t e t h a t t h e exchange o f one a m i n o acid molecule for a n o t h e r on t h e carrier
is faster t h a n the direct b i n d i n g of t h e a m i n o acid to t h e carrier. T h u s one w o u l d
h a v e to assume t h a t t h e i n h i b i t e d a m i n o acid b i n d s t o t h e carrier quite r a p i d l y a n d
t h a t t h e s t i m u l a t e d a m i n o acid b i n d s slowly b u t t h a t t h e exchange r e a c t i o n b e t w e e n
t h e c a r r i e r - a m i n o acid complex a n d free a m i n o acid is r a p i d . JACQUEZ ~ has shown
t h a t a m o d e l of carrier active t r a n s p o r t which i n c o r p o r a t e s these a s s u m p t i o n s can
i n d e e d p r e d i c t t h e p h e n o m e n o n of c o m p e t i t i v e s t i m u l a t i o n . However, w i t h this exp l a n a t i o n one would e x p e c t t h a t for each e x t r a molecule of t h e s t i m u l a t e d a m i n o
a c i d t a k e n u p a molecule of t h e i n h i b i t e d a m i n o acid w o u l d be e x c l u d e d from t h e
carrier. Thus t h i s m o d e l p r e d i c t s a mole-for-mole r e p l a c e m e n t . This difficulty m i g h t
be c i r c u m v e n t e d i f we assume a g r e a t e r n o n - e x c h a n g e efflux r a t e for t h e i n h i b i t e d
t h a n for t h e s t i m u l a t e d a m i n o acid; however, since n o n - e x c h a n g e efflux r a t e s are low
a n d are of t h e s a m e o r d e r of m a g n i t u d e for m o s t a m i n o acids 9 it is u n l i k e l y t h a t this
is a t e n a b l e e x p l a n a t i o n .
Biochim. Biophys. Acta, x35 (z967) 741-75 o
750
J . A . SCHAFER, J. A. JACQUEZ
A postulation of an intracellular action of the inhibited amino acid which is
consistent with current information is found in the explanation of competitive
stimulation offered by 0XENDER AND CHRISTENSEN9. They suggest that the inhibited
amino acid is rapidly accumulated by the non-exchanging 'A-type' carrier for which
the stimulated amino acid has lower affinity, and that once inside, the accumulated
amino acid drives a counterflow of the stimulated amino acid via an exchanging 'Ltype' carrier for which both amino acids have considerable affinity. GUROl~, FANNING
AI~D CHIRIGOS~ have reported obtaining a 50% stimulation of L-tryptophan uptake
by L-phenylalanine. Since both phenylalanine and tryptophan are presumably taken
up primarily by the L system this finding has remained at variance with the suggestion of OXENDER AND CHRISTENSEN. However, we have found no significant stimulation of the uptake of L-tryptophan in the presence of L-phenylalanine (Fig. 4).
It should be observed that any explanation of competitive stimulation which
involves two carriers and exchange diffusion of the intracellular stimulator for extracellular stimulated amino acid would not necessarily imply that the decrease in uptake of the inhibited amino acid must be equal to the increase in stimulated amino
acid uptake. Presumably the exchange via the 'L-type' carrier would be in a I : I
manner. However, if it is also assumed that the stimulated amino acid normally has
a portion of its uptake mediated by the 'A-type' carrier but that competition by the
extracellular inhibited amino acid displaces stimulated amino acid from this carrier,
then the increased uptake of stimulated amino acid would be less than the decreased
uptake of the inhibited amino acid.
A further test of the OXENDER AND CHRISTENSEN proposal 9 is suggested by
the recent investigations of INUI AND CHRISTENSEN 10, who have presented strong
evidence for the existence of the two transport systems (A and L) by showing that
the flux which they attribute to the A system is dependent on extracellular Na+
wheleas that which they attribute to the L system is independent of extracellular
Na +. If true, this implies that the phenomenon of competitive stimulation should
disappear if Na + is omitted from the extracellular medium. The results of experiments to test this are reported in the following paper n.
ACKNOWLEDGEMENTS
This investigation was supported by U.S. Public Health Service Research
Grant No. CA 06734 from the National Cancer Institute.
The authors wish to acknowledge the capable technical assistance of Mr.
J A M E S M. TERRIS.
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2
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io
II
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Biochim. Biophys. Acta, 135 (1967) 741-75o