Planta
Planta 135, 275-283 (1977)
9 by Springer-Verlag 1977
The Specificity of Carrier-mediated Auxin Transport by Suspension-cultured Crown Gall Cells P.H. Rubery Department of Biochemistry, Cambridge University, Tennis Court Road, Cambridge, CB2 1QW, U.K.
1. The specificity of the auxin transport system of suspension-cultured crown gall cells from Parthenocissus tricuspidata Planch- is examined with regard to 2,4-Dichlorophenoxyacetic acid (2,4 D), 1Naphthylacetic acid (NAA) and Benzoic acid (BA) as well as for indole-3-acetic acid (IAA). - 2. All four weak acids can be accumulated by the cells from a medium more acidic than the cytoplasm. This is by virtue of non-specific passive diffusion of their lipid-soluble protonated forms down a concentration gradient. The corresponding anionic species are much less permeant. The extent of the accumulation is dependent on the p H difference that is maintained by the cells between their cytoplasm and the incubation medium. Studies of the concentration dependence of BA and N A A net uptake at a series of external pHs suggest that an acidification of the cytoplasm c a n be eventually brought about by the entry of weak acid into the cells. - 3. The uptake of 2,4 D, as well as that of IAA, has a saturable carrier-mediated component in addition to the passive diffusion of the undissociated acid. These saturable components probably represent anion uptake and appear to be mediated by a c o m m o n carrier. The kinetic studies provided no evidence for the participation of carriers in the transport of BA or NAA. - 4. It was shown that the efflux of 2,4 D also has a carrier-mediated component and it is suggested that both the influx and efflux of I A A and 2,4 D occur on a c o m m o n carrier. - 5. The inhibitor of polar auxin transport, 2,3,5-triiodobenzoic acid (TIBA), stimulates the net uptake of I A A by inhibiting carrier-mediated efflux of I A A from the cells. However, T I B A could not be demonstrated to have a significant effect on 2,4 D transport and any perturbation that occurs is very small in comparison with its effect on I A A movement. Abstract.
Abbreviations: IAA-indol-3-yl acetic acid; 2,4 D=2,4-Dichloro-
phenoxyacetic acid, NAA = 1-Naphthylacetic acid, BA= Benzoic acid, TIBA=2,3,5-triiodobenzoic acid
To account for this, the proposed c o m m o n carrier could exhibit some difference in its internal binding characteristics betweend 2,4 D and IAA. An alternative explanation is that a second carrier is present, which mediates I A A efflux only, and which is inhibited by TIBA. - 6. TIBA has no significant effect on the transport of either BA or NAA, except to bring about an inhibition of net uptake, and a corresponding stimulation of efflux, when it is present at concentrations sufficient to acidify the cytoplasm. 7. The crown gall cells are compared to intact plant tissues capable of polar auxin transport with regard to the specificities exhibited for the transport of the auxins IAA, 2,4 D and N A A and the non-auxin BA. K e y w o r d s : Auxin transport - - Crown gall
nocissus -
Parthe-
Specificity -- Suspension culture.
Introduction
It has been shown previously that I A A is transported across the p l a s m a m e m b r a n e o f P a r t h e n o c i s s u s tricuspidata crown gall suspension culture cells by both diffusion of the lipid-soluble undissociated acid and by a carrier system which mediates the transport of IAA anions. I A A will accumulate in the cells provided that the extracellular p H is below that of the cytoplasm. 2,4 D was shown to be an inhibitor of the carrier-mediated component of I A A uptake whereas T I B A and morphactin inhibited carrier-mediated I A A efflux. A model for polar auxin transport through intact tissues was proposed in which passive carriermediated efflux of auxin anions was suggested to occur preferentially from the basal ends of cells as the result of the assymetric distribution of carrier. It was predicated that this carrier would have similar properties to those described for the system present in the cultured cells (Rubery and Sheldrake, 1973, 1974).
276 The p u r p o s e o f the w o r k d e s c r i b e d in this p a p e r was to e x a m i n e further the specificity o f the auxin t r a n s p o r t system o f the c r o w n gall cells. A p r i n c i p a l a i m was to d e t e r m i n e w h e t h e r 2,4 D u p t a k e has a c a r r i e r - m e d i a t e d c o m p o n e n t a n d if so, w h e t h e r I A A a n d 2,4 D share a c o m m o n carrier. The u p t a k e characteristics o f N A A , which has a u x i n activity, a n d o f b e n z o i c a c i d (BA), which is a lipid-soluble w e a k acid w i t h o u t a u x i n activity, were also investigated. M a n u s c r i p t s in p r e p a r a t i o n will describe the effects o f u n c o u p l e r s a n d c h e m i c a l m o d i f y i n g reagents o n a u x i n t r a n s p o r t a n d will c o n s i d e r the extent to which c a r r i e r - m e d i a t e d auxin t r a n s p o r t is electrically n e u t r a l o r is electrogenic.
P.H. Rubery: Carrier-mediated Auxin Transport
9C 80 ?o co
60
"5 50
~ ~o o
30 20
Materials and Methods
10
[1-14C]IAA ammonium salt (52mCi mmol-1), [1-1~C]2,4D (54 mCi retool- ~), [1-14C]NAA (54 mCi mmol- 1) and [1-14C]BA (60 mCi mmol- 1) were obtained from the Radiochemical Centre, Amersham, Bucks, England. The latter three labelled compounds were supplied as the free acid in benzene solution. Before use, the benzene was removed under vacuum and the solid residue was dissolved in very dilute KHCO3 solution to give a stock solution of 10 gCi ml ~ which was stored frozen. The growth and maintainance of the crown gall cultures has been described previously (Rubery, 1972) as have the incubation conditions and experimental procedures for measurement of uptake of radioactivity (Rubery and Sheldrake, 1973, 1974). In summary: When the time course of Uptake or efflux was followed, it was convenient sequentially t O remove aliquots of cells for measurement of radioactivity from an incubation in a conical flask whose contents were stirred magnetically (designated Method B, Rubery and Sheldrake, 1974). Otherwise, each individual measurement was obtained from a single incubation in a boiling tube kept in a shaking water bath (designated Method A, Rubery and Sheldrake, 1974). All experiments were carried out at 25 C. Uptake was terminated by rapid filtration of the cells followed by weighing and liquid scintillation counting.
0
3.0
410
5'.0 pH
610
7.0
Fig. 1. The relationship between pH and uptake of [1-~4C]2,4 D ( A - ) , [1-14C]BA (-m~ and [1-1~C]NAA (-e-) from 1.0 gmol/1 solutions after 3 rain incubation. Uptake is presented as per-cent of the value at pH 3.0. These values were 850, 1300 and 1600 dpm mg fresh wt 1 respectively
-1,0
"7 -1.5
j/.J "7
=- -2.0 E
@ 7
f
o~ -2.5
Results -3,0
pH-Dependence of the Uptake of [1-14C]2,4 D,-NAA and-BA by Crown Gall Cells The p H - d e p e n d e n c e o f the u p t a k e o f all 3 acids after 3 rain i n c u b a t i o n at an e x t e r n a l c o n c e n t r a t i o n o f 1.0 ~tmol/1 resembles t h a t o f I A A ( R u b e r y a n d Sheldrake, 1973) a n d has the f o r m o f a d i s s o c i a t i o n curve (Fig. 1). The p H values at which 50% o f the maxim u m u p t a k e is o b t a i n e d increase as the strength o f the acid decreases.
The Relationship between External Concentration and Uptake of 2,4 D The u p t a k e o f 2,4 D (0-0.5 retool/l) after 1 rain incub a t i o n was d e t e r m i n e d at p H 4.5, 5.5 a n d 6.5. A
0
0.5
1.0 15 2.0 2.5 log10 [non- radioactive 2,4 D] (,umot/[)
3.0
Fig. 2. The effect of non-radioactive 2,4 D (0-500 ~mol/1) on the uptake of [1-14C]2,4D from 1.0~tmol/1 solutions at pH 4.5 ( A - ) , pH 5.5 ( o - ) and pH 6.5 @l-) after 1 min incubation. Log10 [non-radioactive 2,4D] is plotted versus loglo reciprocal uptake of [1-14C]2,4D. 0.25 gCi [1-~4C]2,4D was present in each incubation
fixed c o n c e n t r a t i o n o f r a d i o a c t i v e 2,4 D was used (1.0 gmol/1), the t o t a l c o n c e n t r a t i o n being v a r i e d by a d d i t i o n o f u n l a b e l l e d 2,4 D. The d a t a m a y be a n a lysed in v a r i o u s ways. F o r instance, the total a m o u n t o f 2,4 D t a k e n up by the cells after 1 min m a y be p l o t t e d as a f u n c t i o n o f 2,4 D c o n c e n t r a t i o n . A l t e r n a tively, n o n - r a d i o a c t i v e 2,4 D m a y be t r e a t e d as a n i n h i b i t o r o f the u p t a k e o f [1-14C]2,4 D a n d it is useful
P.H. Rubery: Carrier-mediated Auxin Transport
277
to plot logl0[non-radioactive 2,4 D] against log~o(uptake [1-1r D rain- 1)- 1 (Fig. 2). This is a double logarithmic version of the conventional Dixon plot (velocity -~ versus inhibitor concentration at fixed substrate c o n c e n t r a t i o n - D i x o n , 1953). It shows the effect of relative changes in inhibitor concentration and allows a wide range of values clearly to be displayed on the Figure. Presentation of the results in this way facilitates comparison with data for BA and NAA (Fig. 3). Convex curves are obtained, indicating that the non-radioactive 2,4 D inhibits most effectively at low concentrations. As the concentration is increased, progressively less inhibition is exerted and a plateau value of uptake of radioactivity is approached. The proportion of the uptake of [1-~4C]2,4 D which is insensitive to inhibition becomes larger as the pH is lowered and at pH 4.5, little inhibition occurs. In other words, the total uptake rate of 2,4 D does not become saturated over the concentration range used. As may be deduced from Figure 2, at pH 5.5 and 6.5, the relationship is non-linear at low 2,4 D concentrations, but becomes linear thereafter (corresponding to the approach to the plateau value in Figure 2). At pH 4.5, departure from linearity at low concentrations is barely apparent. Similar results were previously obtained using IAA (Rubery and Sheldrake, 1973, 1974) which were interpreted in terms of the contribution of a saturable carrier of auxin anions to the total uptake.
The Relationship between External Concentration and Uptake of BA In contrast to the results presented above for 2,4 D, the relationship between total uptake and concentra-7.5
The Effect of Non-radioactive BA on the Time Course of [1-14C]BA Uptake It was necessary to determine whether the inhibition of radioactive BA uptake after 1 rain incubation by unlabelled BA at concentrations exceeding the threshold value was exerted on the initial rate or on the net rate. The uptake of radioactivity from 1.0 ~mol/1 [i-~4C]BA after 15 s and 3 min was measured in the presence and absence of different concentrations of non-radioactive BA. Five replicates were used for each determination and the significance of any difference from the controls was estimated using Student's t test (Table 1). At pH 6.5, 0.67 retool/1 unlabelled BA does not inhibit uptake of radioactivity after 15 s incubation but does inhibit after 3 rain incubation. Greater concentrations inhibit at both times. It appears that at high total concentrations of benzoic acid, a 15 s incubation is an inadequate approxima-
Table I. The effect of non-radioactive BA on the uptake of [1-1 +C]BA from a 1.0 gmol/1 solution at pH 6.5 after 15 s and 3 rain incubation. Values are means _+standard error and numbers of observations are shown in parentheses. Probabilities of difference from the controls incubated without non-radioactive BA are evaluated by Student's t test and shown where not self-evident
s -2.0
"5 E -2.5 E E ca
o~ -3.0
- _3.4 l_ 0.0
tion is linear at low BA concentrations but becomes increasingly curved as the concentration is raised. The data are presented in terms of the inhibition of [114C]BA uptake (1.0 ~tmol/1 external concentration; 1 rain incubation) by non-radioactive BA (0 0.5 retool/l) as described in the previous paragraph (Fig. 3-open symbols). At low concentrations of nonradioactive BA, no inhibition occurs, as indicated by the horizontal regions of the curves. However, at each pH, there appears to be a threshold concentration at which the plots to curve upwards. This-represents the onset of inhibition of uptake of radioactivity. The threshold concentrations become greater as the pH is raised and the content of benzoic acid in the cells after 1 min uptake at these concentrations is approximately 50 pmol/mg fresh wt in each case.
r
i
0.5 1.0 1.'5 2'.0 2.5 tog~0[non-radioactive acid (BA orNAA)] (~mo[/[)
Concentration of non-radioactive BA (mmol/1)
Uptake times
0.0 (45) 0.50 (5) 0.67 (5) 0.83 (5) 1.30 (5) 2.10 (5) 4.20 (5)
13.43 _+0.23 13.60+_ 1.42 (ns) 13.26_+0.17 (ns) 10.48_+0.34 Co
Planta 135, 275-283 (1977)
9 by Springer-Verlag 1977
The Specificity of Carrier-mediated Auxin Transport by Suspension-cultured Crown Gall Cells P.H. Rubery Department of Biochemistry, Cambridge University, Tennis Court Road, Cambridge, CB2 1QW, U.K.
1. The specificity of the auxin transport system of suspension-cultured crown gall cells from Parthenocissus tricuspidata Planch- is examined with regard to 2,4-Dichlorophenoxyacetic acid (2,4 D), 1Naphthylacetic acid (NAA) and Benzoic acid (BA) as well as for indole-3-acetic acid (IAA). - 2. All four weak acids can be accumulated by the cells from a medium more acidic than the cytoplasm. This is by virtue of non-specific passive diffusion of their lipid-soluble protonated forms down a concentration gradient. The corresponding anionic species are much less permeant. The extent of the accumulation is dependent on the p H difference that is maintained by the cells between their cytoplasm and the incubation medium. Studies of the concentration dependence of BA and N A A net uptake at a series of external pHs suggest that an acidification of the cytoplasm c a n be eventually brought about by the entry of weak acid into the cells. - 3. The uptake of 2,4 D, as well as that of IAA, has a saturable carrier-mediated component in addition to the passive diffusion of the undissociated acid. These saturable components probably represent anion uptake and appear to be mediated by a c o m m o n carrier. The kinetic studies provided no evidence for the participation of carriers in the transport of BA or NAA. - 4. It was shown that the efflux of 2,4 D also has a carrier-mediated component and it is suggested that both the influx and efflux of I A A and 2,4 D occur on a c o m m o n carrier. - 5. The inhibitor of polar auxin transport, 2,3,5-triiodobenzoic acid (TIBA), stimulates the net uptake of I A A by inhibiting carrier-mediated efflux of I A A from the cells. However, T I B A could not be demonstrated to have a significant effect on 2,4 D transport and any perturbation that occurs is very small in comparison with its effect on I A A movement. Abstract.
Abbreviations: IAA-indol-3-yl acetic acid; 2,4 D=2,4-Dichloro-
phenoxyacetic acid, NAA = 1-Naphthylacetic acid, BA= Benzoic acid, TIBA=2,3,5-triiodobenzoic acid
To account for this, the proposed c o m m o n carrier could exhibit some difference in its internal binding characteristics betweend 2,4 D and IAA. An alternative explanation is that a second carrier is present, which mediates I A A efflux only, and which is inhibited by TIBA. - 6. TIBA has no significant effect on the transport of either BA or NAA, except to bring about an inhibition of net uptake, and a corresponding stimulation of efflux, when it is present at concentrations sufficient to acidify the cytoplasm. 7. The crown gall cells are compared to intact plant tissues capable of polar auxin transport with regard to the specificities exhibited for the transport of the auxins IAA, 2,4 D and N A A and the non-auxin BA. K e y w o r d s : Auxin transport - - Crown gall
nocissus -
Parthe-
Specificity -- Suspension culture.
Introduction
It has been shown previously that I A A is transported across the p l a s m a m e m b r a n e o f P a r t h e n o c i s s u s tricuspidata crown gall suspension culture cells by both diffusion of the lipid-soluble undissociated acid and by a carrier system which mediates the transport of IAA anions. I A A will accumulate in the cells provided that the extracellular p H is below that of the cytoplasm. 2,4 D was shown to be an inhibitor of the carrier-mediated component of I A A uptake whereas T I B A and morphactin inhibited carrier-mediated I A A efflux. A model for polar auxin transport through intact tissues was proposed in which passive carriermediated efflux of auxin anions was suggested to occur preferentially from the basal ends of cells as the result of the assymetric distribution of carrier. It was predicated that this carrier would have similar properties to those described for the system present in the cultured cells (Rubery and Sheldrake, 1973, 1974).
276 The p u r p o s e o f the w o r k d e s c r i b e d in this p a p e r was to e x a m i n e further the specificity o f the auxin t r a n s p o r t system o f the c r o w n gall cells. A p r i n c i p a l a i m was to d e t e r m i n e w h e t h e r 2,4 D u p t a k e has a c a r r i e r - m e d i a t e d c o m p o n e n t a n d if so, w h e t h e r I A A a n d 2,4 D share a c o m m o n carrier. The u p t a k e characteristics o f N A A , which has a u x i n activity, a n d o f b e n z o i c a c i d (BA), which is a lipid-soluble w e a k acid w i t h o u t a u x i n activity, were also investigated. M a n u s c r i p t s in p r e p a r a t i o n will describe the effects o f u n c o u p l e r s a n d c h e m i c a l m o d i f y i n g reagents o n a u x i n t r a n s p o r t a n d will c o n s i d e r the extent to which c a r r i e r - m e d i a t e d auxin t r a n s p o r t is electrically n e u t r a l o r is electrogenic.
P.H. Rubery: Carrier-mediated Auxin Transport
9C 80 ?o co
60
"5 50
~ ~o o
30 20
Materials and Methods
10
[1-14C]IAA ammonium salt (52mCi mmol-1), [1-1~C]2,4D (54 mCi retool- ~), [1-14C]NAA (54 mCi mmol- 1) and [1-14C]BA (60 mCi mmol- 1) were obtained from the Radiochemical Centre, Amersham, Bucks, England. The latter three labelled compounds were supplied as the free acid in benzene solution. Before use, the benzene was removed under vacuum and the solid residue was dissolved in very dilute KHCO3 solution to give a stock solution of 10 gCi ml ~ which was stored frozen. The growth and maintainance of the crown gall cultures has been described previously (Rubery, 1972) as have the incubation conditions and experimental procedures for measurement of uptake of radioactivity (Rubery and Sheldrake, 1973, 1974). In summary: When the time course of Uptake or efflux was followed, it was convenient sequentially t O remove aliquots of cells for measurement of radioactivity from an incubation in a conical flask whose contents were stirred magnetically (designated Method B, Rubery and Sheldrake, 1974). Otherwise, each individual measurement was obtained from a single incubation in a boiling tube kept in a shaking water bath (designated Method A, Rubery and Sheldrake, 1974). All experiments were carried out at 25 C. Uptake was terminated by rapid filtration of the cells followed by weighing and liquid scintillation counting.
0
3.0
410
5'.0 pH
610
7.0
Fig. 1. The relationship between pH and uptake of [1-~4C]2,4 D ( A - ) , [1-14C]BA (-m~ and [1-1~C]NAA (-e-) from 1.0 gmol/1 solutions after 3 rain incubation. Uptake is presented as per-cent of the value at pH 3.0. These values were 850, 1300 and 1600 dpm mg fresh wt 1 respectively
-1,0
"7 -1.5
j/.J "7
=- -2.0 E
@ 7
f
o~ -2.5
Results -3,0
pH-Dependence of the Uptake of [1-14C]2,4 D,-NAA and-BA by Crown Gall Cells The p H - d e p e n d e n c e o f the u p t a k e o f all 3 acids after 3 rain i n c u b a t i o n at an e x t e r n a l c o n c e n t r a t i o n o f 1.0 ~tmol/1 resembles t h a t o f I A A ( R u b e r y a n d Sheldrake, 1973) a n d has the f o r m o f a d i s s o c i a t i o n curve (Fig. 1). The p H values at which 50% o f the maxim u m u p t a k e is o b t a i n e d increase as the strength o f the acid decreases.
The Relationship between External Concentration and Uptake of 2,4 D The u p t a k e o f 2,4 D (0-0.5 retool/l) after 1 rain incub a t i o n was d e t e r m i n e d at p H 4.5, 5.5 a n d 6.5. A
0
0.5
1.0 15 2.0 2.5 log10 [non- radioactive 2,4 D] (,umot/[)
3.0
Fig. 2. The effect of non-radioactive 2,4 D (0-500 ~mol/1) on the uptake of [1-14C]2,4D from 1.0~tmol/1 solutions at pH 4.5 ( A - ) , pH 5.5 ( o - ) and pH 6.5 @l-) after 1 min incubation. Log10 [non-radioactive 2,4D] is plotted versus loglo reciprocal uptake of [1-14C]2,4D. 0.25 gCi [1-~4C]2,4D was present in each incubation
fixed c o n c e n t r a t i o n o f r a d i o a c t i v e 2,4 D was used (1.0 gmol/1), the t o t a l c o n c e n t r a t i o n being v a r i e d by a d d i t i o n o f u n l a b e l l e d 2,4 D. The d a t a m a y be a n a lysed in v a r i o u s ways. F o r instance, the total a m o u n t o f 2,4 D t a k e n up by the cells after 1 min m a y be p l o t t e d as a f u n c t i o n o f 2,4 D c o n c e n t r a t i o n . A l t e r n a tively, n o n - r a d i o a c t i v e 2,4 D m a y be t r e a t e d as a n i n h i b i t o r o f the u p t a k e o f [1-14C]2,4 D a n d it is useful
P.H. Rubery: Carrier-mediated Auxin Transport
277
to plot logl0[non-radioactive 2,4 D] against log~o(uptake [1-1r D rain- 1)- 1 (Fig. 2). This is a double logarithmic version of the conventional Dixon plot (velocity -~ versus inhibitor concentration at fixed substrate c o n c e n t r a t i o n - D i x o n , 1953). It shows the effect of relative changes in inhibitor concentration and allows a wide range of values clearly to be displayed on the Figure. Presentation of the results in this way facilitates comparison with data for BA and NAA (Fig. 3). Convex curves are obtained, indicating that the non-radioactive 2,4 D inhibits most effectively at low concentrations. As the concentration is increased, progressively less inhibition is exerted and a plateau value of uptake of radioactivity is approached. The proportion of the uptake of [1-~4C]2,4 D which is insensitive to inhibition becomes larger as the pH is lowered and at pH 4.5, little inhibition occurs. In other words, the total uptake rate of 2,4 D does not become saturated over the concentration range used. As may be deduced from Figure 2, at pH 5.5 and 6.5, the relationship is non-linear at low 2,4 D concentrations, but becomes linear thereafter (corresponding to the approach to the plateau value in Figure 2). At pH 4.5, departure from linearity at low concentrations is barely apparent. Similar results were previously obtained using IAA (Rubery and Sheldrake, 1973, 1974) which were interpreted in terms of the contribution of a saturable carrier of auxin anions to the total uptake.
The Relationship between External Concentration and Uptake of BA In contrast to the results presented above for 2,4 D, the relationship between total uptake and concentra-7.5
The Effect of Non-radioactive BA on the Time Course of [1-14C]BA Uptake It was necessary to determine whether the inhibition of radioactive BA uptake after 1 rain incubation by unlabelled BA at concentrations exceeding the threshold value was exerted on the initial rate or on the net rate. The uptake of radioactivity from 1.0 ~mol/1 [i-~4C]BA after 15 s and 3 min was measured in the presence and absence of different concentrations of non-radioactive BA. Five replicates were used for each determination and the significance of any difference from the controls was estimated using Student's t test (Table 1). At pH 6.5, 0.67 retool/1 unlabelled BA does not inhibit uptake of radioactivity after 15 s incubation but does inhibit after 3 rain incubation. Greater concentrations inhibit at both times. It appears that at high total concentrations of benzoic acid, a 15 s incubation is an inadequate approxima-
Table I. The effect of non-radioactive BA on the uptake of [1-1 +C]BA from a 1.0 gmol/1 solution at pH 6.5 after 15 s and 3 rain incubation. Values are means _+standard error and numbers of observations are shown in parentheses. Probabilities of difference from the controls incubated without non-radioactive BA are evaluated by Student's t test and shown where not self-evident
s -2.0
"5 E -2.5 E E ca
o~ -3.0
- _3.4 l_ 0.0
tion is linear at low BA concentrations but becomes increasingly curved as the concentration is raised. The data are presented in terms of the inhibition of [114C]BA uptake (1.0 ~tmol/1 external concentration; 1 rain incubation) by non-radioactive BA (0 0.5 retool/l) as described in the previous paragraph (Fig. 3-open symbols). At low concentrations of nonradioactive BA, no inhibition occurs, as indicated by the horizontal regions of the curves. However, at each pH, there appears to be a threshold concentration at which the plots to curve upwards. This-represents the onset of inhibition of uptake of radioactivity. The threshold concentrations become greater as the pH is raised and the content of benzoic acid in the cells after 1 min uptake at these concentrations is approximately 50 pmol/mg fresh wt in each case.
r
i
0.5 1.0 1.'5 2'.0 2.5 tog~0[non-radioactive acid (BA orNAA)] (~mo[/[)
Concentration of non-radioactive BA (mmol/1)
Uptake times
0.0 (45) 0.50 (5) 0.67 (5) 0.83 (5) 1.30 (5) 2.10 (5) 4.20 (5)
13.43 _+0.23 13.60+_ 1.42 (ns) 13.26_+0.17 (ns) 10.48_+0.34 Co
