Mar 25, 1970 - East Rutherford, N.J.). The samples were miscible with the p-dioxane, eliminating any need for evapora- tion, without decreasing counting ...
JOURNAL OF BACTERIOLOGY, July 1970, p. 120-130 Copyright a 1970 American Society for Microbiology
Vol. 103, No. I Printed in U.S.A.
Amino Acid Transport by the Filamentous Fungus Arthrobotrys conoides' RISHAB K. GUPTA2 AND DAVID PRAMER Department ofBiochemistry and Microbiology, Rutgers-The State University, New Brunswick, New Jersey, 08903 Received for publication 25 March 1970
Uptake of L-valine by germinated spores of Arthrobotrys conoides has all the characteristics of a system of transport that requires an expenditure of energy by the cells. It is dependent on temperature and has an energy of activation of 16,000 cal/mole. Uptake is optimal at pH 5 to 6. L-Valine accumulated against a concentration gradient and is not lost from the cells by leakage or exchange. The process requires energy supplied by the metabolic reactions that are inhibited by catalytic amounts of 2, 4-dinitrophenol and azide. The kinetics of the system are consistent with a mechanism of transport that depends on a limited number of sites on the cell surface, and the Michaelis constant for the system is 1.5 X 10-5 to 7.5 X 10-5 M. Modification of the amino or carboxyl group abolishes L-valine uptake. The process is competitively inhibited by D-valine, glycine, and other neutral amino acids (Ki 1.5 x 10-5 to 4.0 X 10-5 M), indicating a lack of stereospecificity, and also indicating that aliphatic side chain is not required for binding with the carrier. The transport system has less affinity for acidic amino acids (glutamic and aspartic acids) than neutral amino acids, and a greater affinity for basic amino acids (histidine, lysine, and arginine). The range of affinity is in the order of 100, as measured in terms of Ki values for various compounds. The data presented provide suggestive evidence that the uptake by A. conoides of all amino acids except proline is mediated by a single carrier system that possesses an overall negative charge. =
Nematode-trapping fungi are fascinating organisms that use specialized structures to trap, kill, and digest their prey (15). Traps are not always formed by these fungi unless nematodes are in their vicinity. Nematodes seem to trigger the formation of traps by producing a morphogenetic substance which was named "nemin" (17). Chemical fractionation of nematodes suggested that nemin is a peptide-like substance (16). Some commercially available amino acids, particularly the branched-chain, aliphatic compounds, valine, leucine, and isoleucine, were found to have nemin activity and, of these, valine was the most effective (L. M. Wootton and D. Pramer, Bacteriol. Proc., p. 75, 1966). When an attempt was made to explain the activity of valine by reference to the literature, it became evident that little was known of amino acid uptake and metabolism by filamentous fungi. Most work in this area has been done with animal cells and tissues, and bacteria. The
only fungi which have been studied with any intensity are yeast cells, and amino acid uptake by filamentous fungi remains practically unexplored. Therefore, studies of valine uptake by the nematode-trapping fungus Arthrobotrys conoides were performed to identify the mechanism by which the amino acid enters fungal cells and to characterize the site on the cell surface that controls the transport phenomenon.
MATERIALS AND METHODS Organism. The nematode-trapping fungus used throughout the present investigation was A. conoides. The culture was originally isolated from decomposing leaves by one of the authors (D. Pramer) and identified by C. Drechsler. It is Deuteromycete, included in the order Moniliales and the family Moniliaceae. Preparation of cells for uptake of valine. Preliminary tests indicated that germinated spores were ideally suited for the present purpose, so a series of experiments was performed to define conditions that would yield large quantities of consistently uniform germinated spores for use in studies of valine uptake and 1 Paper of the Journal Series, New Jersey Agricultural Experimetabolism. Germinated spores have been employed ment Station, New Brunswick, N.J. in studies of the uptake of tryptophan and uridine 2 Present address: Department of Medical Microbiology and Immunology, School of Medicine, University of California, by Neurospora crassa (20). Plates of corn meal extract-agar (Difco Laboratories, Detroit, Mich.) Los Angeles, Calif. 90024. 120
VOL. 103, 1970
AMINO ACID TRANSPORT BY A. CONOIDES
were inoculated with spores of A. conoides and incubated at 28 C for 15 to 18 days. The surface of each plate was flooded with 5 ml of sterile 0.02 M phosphate buffer (pH 6), and spores were suspended in the buffer by gentle rubbing with a steam-sterilized policeman. The surface of each plate was washed twice. The harvest from a large number of plates was combined in a 500-ml sterile Erlenmeyer flask and agitated vigorously to free conidia from conidiophores. The suspension was then filtered through sterile multilayered cheese cloth. Filaments were retained on the cloth and spores were collected with the filtrate. The spores were concentrated by centrifugation for 5 min at 2,200 X g, washed twice, suspended in 0.02 M phosphate buffer (pH 6), and stored at 0 to 4 C until used. Storage seldom exceeded a period of 4 hr. The spores were germinated in a solution containing 1% glucose and 0.5% corn meal extract (Colab Laboratories, Inc., Chicago Heights, Ill.) in 0.02 M phosphate buffer at pH 6. The solution was prepared at double strength and received an equal volume of spore suspension that was previously diluted to contain 4 X 106 spores/ml. The final suspension had a volume of 40 ml in 250-ml Erlenmeyer flasks, and a spore density in the order of 2 X 106 spores/ml. Flasks were incubated on a rotary shaker at 28 C for 8 hr. Under these conditions spore germination was
90%.
Germinated spores were collected by centrifugation at 2,200 X g for 5 min at room temperature, washed three times with cold sterile 0.02 M phosphate buffer (pH 6), resuspended in the same buffer, and stored in ice until used. To facilitate the preparation of washed germinated spore suspensions (hereafter referred to as cell suspension) of uniform and reproducible density, a calibration curve was prepared relating turbidity and dry weight. Density of cells in the suspension was measured in a Klett-Summerson Spectrophotometer with a red filter (No. 66). From the relation between Klett units and dry weight of germinated spores of A. conoides, it was possible to dilute the cell suspensions rapidly to densities required for any experimental purpose. Measurement of L-valine uptake. Throughout these studies, uniformly labeled 14C-L-valine was used. It was purchased from New England Nuclear Corp., Boston, Mass., and had a specific activity of 190 mcj mmole. The compound was checked for purity by silica gel thin-layer chromatography in butanolacetic acid-water (8:2:8). Only one spot corresponding to valine appeared when the plates were developed by autoradiography. Equal volumes of double-strength radioactive valine and cell suspension prepared in 0.02 M phosphate buffer and were placed in separate 50-ml beakers in a water-bath shaker (New Brunswick Scientific Co.) at 28 C for 10 min. After temperature equilibration, the valine solution and cell suspension were rapidly mixed. A sample of approximately 1.0 ml was withdrawn immediately and filtered through a membrane filter with a pore diameter of 0.45 .rm (Millipore Corp., Bedford, Mass.). This process took about 3
121
to 4 sec. Filtrations were performed by using an assembly of 10 units connected to a vacuum pump through a manifold with stopcocks for each individual unit. The stopcock was opened and vacuum was applied before the membrane received the sample, so that cells and solution were separated as quickly as possible after being added to a filter unit. A rimless tube (15 by 85 cm) received the filtrate. Samples were withdrawn and filtered by these procedures at intervals that were appropriate or required by experimental
design. Unless described otherwise, L-valine uptake by A. conoides was measured under the following standardized conditions. A suspension of freshly germinated spores was placed in a beaker and treated with a solution of "4C-L-valine, so that the final preparation contained 2 X 10-5 M 14C-L-valine and 0.5 mg (dry weight) of cells/ml of 0.02 M phosphate buffer at pH 6. Measurement of radioactivity. Portions of the filtrate (0.1 ml) for analysis were placed in vials, and each was diluted with 10 ml of scintillation liquid that consisted of 7 g of 2, 5-diphenyloxazol, 0.05 g of 1,4bis-2 (5-phenyloxazolyl) benzene (Nuclear Associates, Inc., Westbury, N.Y.), and 70 g of naphthalene in 1,000 ml of p-dioxane (Matheson, Coleman, and Bell, East Rutherford, N.J.). The samples were miscible with the p-dioxane, eliminating any need for evaporation, without decreasing counting efficiency due to quenching (3). The radioactivity of samples was measured for a minimum of 10 min in a liquid scintillation counter (Nuclear Chicago Corp., Des Plaines, Ill.; system 6804). All counts were corrected for background. In some experiments cells were fractionated and their contents were analyzed for radioactivity. Since it was anticipated that cell extracts would affect the efficiency of measurements, the counts were corrected for the quenching. RESULTS
Time course of valine uptake. The time course of L-valine uptake by A. conoides is shown in Fig. 1. L-Valine uptake began immediately after the addition of the amino acid to the cell suspension, and accumulation of L-valine continued as a linear function of time until the supply in the medium was nearly exhausted. Most of the amino acid was accumulated by the cells from the medium within 4 to 6 min. To determine the ability of A. conoides to retain absorbed L-valine, cells were treated with the 'IC-amino acid for 10 min under conditions that favored uptake. They were then washed twice with cold 0.02 M phosphate buffer at pH 6, by using a refrigerated centrifuge, and tests showed that no radioactivity was lost by the cells to the washes. Finally, the '4C-L-valine-loaded cells were divided into two equal portions. One was suspended in buffer containing unlabeled Lvaline (2 X 10-4 M) and the other was suspended in buffer only. Both of these suspensions were
122
GUPTA AND PRAMER
J. BACTERIOL.
, . , drawn for analysis. One-half of each sample was .___v___.____used to measure 14C-L-valine uptake. The remaining 1.0 ml was added to an equal volume of
cold 10% trichloroacetic acid and extracted for 30 min. Cold acid-soluble and insoluble materials
U
acb.
were then separated by filtration and tested for radioactivity. By this procedure it was possible to trace the movement of L-valine from solution n0 into cell, and determine whether intracellular L'0 valine remained free (cold acid-soluble) or was incorporated into macromolecules (acid-insoluble). z From Fig. 3, which illustrates the results of this experiment, it was immediately apparent that, binitially, all absorbed L-valine was recovered by IL extraction with cold trichloroacetic acid but, after 5 min, some of the amino acid was incorporated into the macromolecular fraction of cells. The concentration of free L-valine then decreased i (cold acid-soluble) and that of cold acid-insoluble 0 2 3 4 S 1 6 7 8 9 L-valine increased. This trend was continuing MINUTES when uptake of L-valine from the external soluFIG. 1. Time course of L-valinlre uptake by A. tion had ceased, and the experiment was termi0.5 mg (dry conoides at 28 C. The system conte a major portion of L-valine that weight) of cells and 2 X 10-5 ,umole. Of L-valine/ml Of nated. Since entered cells did so before any was incorporated 0.02 M phosphate buffer at pH 6. into macromolecules, the force driving amino placed in a water-bath shaker at 28 C and tested acid uptake by A. conoides was not protein at 2-min intervals for release of radioactivity. synthesis. Thin-layer chromatography and autoradiography were used to identify the "4C acThe experiment was terminated after 18 min. The capacity of loaded cells to retain the amino tivity of cold trichloroacetic acid extracts as Lacid was impressive (Fig. 2). A low order of valine. Zalokar (22) reported a similar behavior radioactivity was present in solultion immediately of proline uptake and incorporation in N. crassa. Influence of temperature and pH on uptake of after the cells were suspended, arid the magnitude of this initial release did not differ significantly with treatment. The amount of radioactivity in 26 solution tended to increase with time, but the 24 change was not great and, at the very most, 8% of the absorbed '4C-L-valine was released by the 201 cells in 18 min. The presence of unlabeled Lvaline did not affect the release of radioactivity. no u 16 The buffered L-valine solution had 1,220 counts per min per ml, and the buffer alone had 820 "O12 counts per min per ml when the experiment was terminated; it is doubtful that this difference is
ainfed
significant. From these results, it appeared that the primary movement of L-valine was in one direction only. The amino acid was accumulated and retained by cells of A. conoides against a concentration gradient, and internal labeled L-valine did not exchange with external L-valine to any significant extent. Fate of absorbed L-valine. To determine the fate of intracellular '4C-L-valine, A. conoides was supplied with the amino acid under standard conditions. At 1.0-min intervals for a total of 12 min, 2-ml portions of the suspension were with-
-12
-3
-4
0
0
4
*
12
16
20
MINUTES
FIG. 2. Accumulationt of '4C-L-valine and release of accumulated radioactivity by A. conoides cells. 0, Accumulation of '4C-L-valine; X, release of 14Cactivity by cells in 0.02 M phosphate buffer (pH 6) containinig 2 X 10-4 M "C-L-valine; A, release of '4C-L-valine by cells in 0.02 M phosphate buffer (pH 6) only. A gap in abcissa represents the time required for transfer of cells from the labeled to nonlabeled medium.
8/~ CO
VOL. 103, 1970
L-valine. The dependence of L-valine uptake
on
temperature was investigated, and the data were used to calculate an energy of activation and temperature coefficient for the system. The temperature was varied from 0 to 45 C in increments of 4 + 0.5 C. Equal volumes of solution
of L-valine (4 x 10-5 M) and a cell suspension (2 mg, dry weight per ml) equilibrated with the test temperature were mixed and maintained at that temperature. At temperatures above 16 C, most L-valine was accumulated by the cells within 10 min. At temperatures below 16 C, the extent of L-valine uptake remained low and never equalled that attained at higher temperatures within the 30-min experimental period. In Fig. 4, the rate of L-valine uptake during the initial 1.0-min period of time is plotted against temperature. Rate increased with increasing temperature to a maximum at 32 C. Further increases in temperature resulted in a rapid decrease in the rate of L-valine uptake. The energy of activation of L-valine uptake was estimated graphically and also computed to be 16,054 cal/mole and 15,977 cal/mole, respectively, between temperatures of 16 and 32 C. These values agree well with each other. A Qlo value of 2.4 was calculated for Lvaline uptake with temperatures of 20 and 30 C. Separate portions of cells of A. conoides were washed and then suspended in solutions that varied from pH 3 to 9. A series of solutions of Lvaline were prepared at these same reactions and were mixed with equal volumes of the appropriate cell suspensions. In every case, solutions were adjusted to the desired pH with HCl or NaOH, and the final test system contained 0.5 mg cells/ ml and 2 X 10-5 M "C-L-valine. Uptake was measured at intervals for 5 min at 28 C. The pH of each suspension was determined at the termina-
s
32
24
_ 16
z
123
AMINO ACID TRANSPORT BY A. CONOIDES
COLD
tion of the experiment and was found unchanged. The results plotted in Fig. 5 indicate an optimum of pH 5 to 6 for L-valine uptake. The system was more sensitive to increased than to decreased hydrogen-ion concentration around the optimum: uptake was diminished more rapidly at pH 4 and 3 than at pH 7 to 9. Dependence of L-valine uptake on metabolic activity. The foregoing studies indicated that the
z
l
£ 0
>
FIG. 4. Influence of tempercature on the rate of Lvali/e uptake by A. conoides at pH 6.
'z i
_K
12
£ IE 0
es 0
8
I-
4 z 0
4
TCA-INSOLUSLE
MINUTES
FIG. 3. Uptake and incorporation of L-valine by A. conoides.
3
4
5
6
7
8
9
pH
FIG. 5. Influence of pH on} the rate of L-valine uptake by A. conoides at 28 C.
124
L-valine uptake by A. conoides was dependent on pH and temperature. It had an energy of activation that equalled that of enzymatic reactions, and was unidirectional. It remained to determine whether the uptake of L-valine by A. conoides required an expenditure of energy by the cell. For this purpose, use was made of the metabolic inhibitors, 2,4-dinitrophenol and sodium azide. Cell suspensions (2 mg/ml) were treated at 28 C for 15 min with 2,4-dinitrophenol at concentrations ranging from 10-2 to 10-5 M. They then received "4C-L-valine (10-4 M), and amino acid uptake was measured after 2.5 min. Oxygen uptake by cell suspensions with and without added 2,4-dinitrophenol was also measured by using a microrespirometer (Medical Electronics, Inc., Middleton, Wis.). It is evident from the results listed in Table 1 that 2,4-dinitrophenol is an inhibitor of L-valine uptake. The extent of the inhibition varied directly with concentration and was approximately 50% at a 2,4-dinitrophenol level of 10-4 M. Since cellular respiration was not inhibited by the concentration of 2,4-dinitrophenol that interfered with amino acid uptake, it was concluded that the effect was specific and not due to death of the cells. 2,4-Dinitrophenol deprives the cell of energy by uncoupling oxidative phosphorylation from the respiratory chain. Apparently, cellular energy was required for the uptake of L-valine, and respiration in presence of 2, 4-dinitrophenol was increased due to loss of respiratory control. The effect of sodium azide on uptake of Lvaline by A. conoides was studied at concentrations ranging from 10-2 to 10-5 M, by using the same procedure employed in testing 2,4-dinitrophenol. Results (Table 2) indicate that sodium azide blocked the uptake of L-valine, and the inhibition increased with an increase in inhibitor TABLE 1. Influence of 2,4-dinitrophenol L-valine and oxygen uptake
on
L-Valine uptake
Oxygen uptakeb
Inhibitor
Activitya
0
10.4
100
61
7.6 5.6 2.2 0.5
73 54 21 5
66 91 117 173
10-3 10-2
b
Expressed Expressed
hour.
control
of
1o-6 10-4
a
J . BACTER IOL.
GUPTA AND PRAMER
as as
10-3 Jmole
per mg per min. per milligram per
microliters
TABLE 2. Influence of sodium azide on L-valine uptake L-Valine uptake Inhibitor Activit? Activitya
0 10-5
104 10-3 10-2 a
10.4 8.7 5.2 1.6 0.4
cent ~ofPercontrol
100 83 50 15 4
Expressed as 10-3 Amole per mg per min.
concentration. Quantitatively the effect was similar to that obtained with 2,4-dinitrophenol. The 50% inhibition observed at 10-4 M concentrations of azide and 2,4-dinitrophenol provided evidence of an energy requirement for L-valine uptake by A. conoides. Kinetics of L-valine uptake. To obtain a Michaelis-Menten constant (Kin) for L-valine transport by A. conoides, the influence of concentration on the rate of uptake of the amino acid was investigated over the range 10-5 to 7.5 X 10-4 M. A plot of the results yielded a typical saturation curve (Fig. 6), indicating that L-valine uptake was limited to a finite number of sites at the cell sui fLice: the carrier system was of limited capacity. Figure 7 is a Lineweaver-Burk (12) double reciprocal plot of the data presented in Fig. 6. Linearity here is indicative that amino acid transport, as enzyme reactions, involved the formation and dissociation of an intermediate complex. Since the curve did not incline towards the origin, diffusion or passive transport did not influence the accumulation of L-valine by A. conoides over the concentration range tested (1, 4). Figures 6 and 7 are typical of the results of seven independent experiments that yielded Km values for the L-valine transport system varying from 1.5 x 10-5 to 7.5 x 10-s M. The affinity of the amino acid for the carrier site is apparently great. Characterization of carrier-site involved in the uptake of L-valine. The inhibition by D-valine of L-valine uptake was measured under standard conditions. The L-isomer was used at levels ranging from 2 X 10-5 to 10-4 M in the presence and absence of D-valine concentrations of 2 X 10-5 and 5 x 10-5 M. A double-reciprocal plot (12) of the results is illustrated by Fig. 8. The three lines represent L-valine uptake in the absence of D-valine and in the presence of Dvaline at each of the two different concentrations. There was a decrease in uptake (increase
in 1 /v) as the concentration of D-valine was increased. All three lines had a common intercept on the vertical axis that corresponded to the maximum velocity of uptake when carrier sites were saturated, and indicated that D-valine competed with L-valine for transport. By using the intercepts in Fig. 8, Km and Vnmax values for L-valine uptake were determined to be 3.7 X 10-5 M and 17.5 x 10-3 ,moles per mg per min, respectively. The Ki values for D-valine were calculated to be 4 X 1O-- and 4.8 X 10-5 M when the amino acid was present at levels of 5 X 10-5 and 2 X 10-5 M, respectively. It is interesting to note that the Ki of D-valine (4 X 10-5 M) and the Kn of L-valine (3.7> X 10-5 M) were almost identical, indicating that the site of valine uptake had equal affinity for the D- and L-isomer, and that the A. conoides transport system lacked stereospecificity. A similar absence of stereospecificity was observed by Yoder et al. (21) in studies of the uptake of amino acids and dipeptides by Leuconostoc mesenteroides. They reported both isomers of valine were rapidly transported and accumulated by the bacterium. Tests of L-valine methyl ester, glycine methyl ester, histamine, or isobutylamine failed to reveal any influence on the uptake of L-valine (Table 3). The last named compounds lack the a-carboxyl group of histidine and valine, respectively. The methyl ester of glycine was of interest because the parent compound, glycine, interfered with Lvaline uptake. From this absence of activity, it was evident that compounds without a functional a-carboxyl group did not compete with L-valine, that the carrier site was group-specific, and that an a-carboxyl group was essential for L-valine transport. Since uptake was decreased by decreasing pH below 5 (Fig. 5), the a-carboxyl groups should be unprotonated or the amino
am
1 12
>
N
1a.
.
*
w
4
10
10
125
AMINO ACID TRANSPORT BY A. CONOIDES
VOL. 103, 1970
20
30
40
0o
L-VALI FIG. 6. Influence of concentration NE
L-valine uptake by A. conoides.
60
70
10
on
the
rate
-2 104
L-VALINE (M)
FIG. 7. Lineweaver-Burk reciprocal plot for L-valine uptake. V = Micromoles of L-valine uptake per mg (dry weight) per min.
acid will not have maximal possible affinity for its binding site. To determine if the a-hydrogen of L-valine was essential for uptake, the effect of a-aminoisobutyric acid was measured at levels of 2 X 10-5 M, 4 X 10-5 M, and 8 X 10-5 M. L-Valine was used at a concentration of 2 x 10-5 M through these studies. Since a-amino-isobutyric acid has no a-hydrogen, it would competitively inhibit Lvaline uptake only if the binding site lacked specificity for the relevant group. This was observed to be the case, and in presence of aaminoisobutyric acid the uptake of L-valine was decreased; the decrease was proportional to the concentration of inhibitor. That the inhibition was competitive in nature was evident from the linear relation between L-valine uptake and inhibitor concentration (Fig. 9). The point of intersection of this line with one horizontal extended left of the vertical at a height of I /Vmax was equal to -Ki (6). The Ki value for aaminoisobutyric acid was 4.2 X 10-5 M. It did not differ significantly from the average Km value (4 X 10-S M) for L-valine, indicating that both compounds had equal affinity for the site of amino acid transport by A. conoides, and that there existed no requirement by that site for the ahydrogen of L-valine. The results listed in Table 4 show that none of four compounds lacking a free a-amino group significantly affected the rate of uptake of L-valine. N-monomethyl DL-valine had a positively charged
J. BACTERIOL.
GUPTA AND PRAMER
126
2
-1
-2
-3
l4 L-VALINE (M)
FIG. 8. Lineweaver-Burk reciprocal plot of L-valinie uptake in the presence and in the absenice of D-valine. Symbols: 0, S X 10-5 M D-valine presentt; A, 2 X 10-5 1 D-valilie present; *, D-valine absent. V = Micromoles of L-valine uptake per mg (dry weiglht) per miii.
TABLE 3. Influence of various compounids that lack or have a modified a-carboxyl group on the uptake of L-valine (1Concn (lOs5 m)
Test compound Test cmpound
None (control) L-Valine methyl ester
L-Valine Per
Glycine methyl ester Isobutylamine Histamine
cent
Test compound
of
uptakea
control
5.0 4.9 5.1 5.1 4.9 4.6 5.1 5.8 6.1
100 99 102 102 98 93 10 115 122
4 8 4 8 4 8 4 8
TABLE 4. Influenice of various compouiids that lack or have a modified a-aminio group oii the uptake of L-valine
a The test compounds were added simultaneously with L-valine (2 X 10-5 M) to the cell suspension, and uptake of the amino acid was measured as 10-3 Mmole of L-valine per mg (dry weight of cells) per min.
Concn L-Valine Perofcent (10-5 m) uptakea control
None (control)
N-monomethyl DL-valine
Proline
a-Ketoisovaleric acid Butyric acid
2 4 10 20 2 4 8 2 4 8 2 4 8
4.90 4.96 4.96 5.00 5.00 4.44 5.12 4.96 5.08 4.80 4.82 5.14 4.90 4.96
100 101 101 102
102 90 104 101 103 97 98
106 100
101
a The test compounds were added simultaneously with L-valine (2 X 10-5 M) to the cell suspen-
sion, and uptake of the amino acid was measured as 10-3 JAmole of L-valine per mg (dry weight of cells) per min.
_*
-4
_2
O
2
a-AMINOISOSUTIRIC
4 ACID
6 10
FIG. 9. Graphic determination of Ki for ce-aminoisobutyric acid. V = Micromoles of L-valine uptake per mg (dry weight) per miii.
secondary amino group under the experimental conditions (pH 6), but it did not compete for the carrier site, possibly owing to steric effects resulting from the bulky nature of the added methyl group. The configuration of proline differs greatly from that of valine and it has a modified amino group. a-Ketoisovaleric acid, the product of valine deamination, and butyric acid were also tested for their ability to inhibit the L-valine up-
AMINO ACID TRANSPORT BY A. CONOIDES
VOL. 103, 1970
take because they lack the a-amino group. From these observations it was apparent that an aamino group is an absolute requirement for uptake of a compound by the A. conoides carrier system responsible for L-valine transport. ,B-Alanine and -y-aminobutyric acid were both observed to interfere with the uptake of L-valine. Subsequent results indicated that fl-alanine had a Ki value of 3.7 X 10-5 M and the Ki for L-alanine was 1.8 X 10-5 M, indicating that the latter compound had a greater affinity than the former for the site of amino acid uptake by A. conoides. -y-Aminobutyric acid, with a greater displaced amino group, had a Ki of 8 X 10-4 M. The magnitude of this value reflects the poor affinity of ,y-aminobutyric acid for the L-valine carrier site. Apparently displacement of the amino groups from the a-carbon can be tolerated only within certain limits if the geometric requirements of the carrier are to be satisfied by the amino and carboxyl groups. L-Leucine and L-isoleucine are structurally related to L-valine, and have been described by many different workers as able to inhibit the uptake of L-valine (5, 7). They and the following other neutral amino acids were tested for an influence on the A. conoides system of L-valine transport: glycine, L-alanine, L-phenylalanine, L-methionine, DL-a-aminoisobutyric acid, Lserine, and L-cysteine. Graphic procedures were used to obtain Ki values for the amino acids. The Km value obtained in these analyses varied from 1.5 X 10-5 to 7.5 X 10-5 M. The Ki values for L-leucine (Fig. 10) and L-isoleucine (Fig. 11) were 1.6 X 10-5 and 2.0 X 10-5 M, respectively.
-6
.4
.2
0
127
2
L- ISOL E UC I NE
4
6
8
10
10 M
FIG. 11. Graphic determination ofKfor L-isoleucine. Symbols: *, 1.5 X 10J4 M L-valinte; A, 7.5 X 10J5 M L-valine; 0, S X 10-J M L-valine; A, 2 X 10-J M L-valine. V = Micromoles of L-valine uptake per mg (dry weight) per min.
L-Leucine and L-isoleucine were competitive inhibitors of L-valine uptake and each had an affinity for the carrier site that was equal to that of L-valine. Generally this was true also for the other compounds of this series. The Ki values obtained for glycine, L-alanine, L-phenylalanine, L-methionine, DL-a-aminoisobutyric acid, Lserine, and L-cysteine are listed in Table 5. These values were almost equal to the Ki value for Lvaline uptake. Therefore, neutral amino acids, irrespective of the nature and length of side chain, were equally active in competing with L-valine for uptake by A. conoides. In general, three amino acid transport systems are believed to function in most cells, one each for neutral, acidic, and basic amino acids. Since a single site was concerned with neutral compounds in A. conoides, two acidic amino acids, L-glutamic and L-aspartic acids, were tested to determine whether they compete with L-valine or are transported by a second system. The results of these tests established that Laspartic acid and L-glutamic acid had Ki values of 3.8 X 10-4 M and 5.0 X 10-4 M, respectively (Table 5). These values are about 10 times greater than the Km, value for L-valine uptake. The acidic amino acids competed with L-valine but had less affinity for the binding site. Reduced affinity may have resulted from the negative change -4 -2 2 4 10 6 a present on the side-chain carboxyl group of acidic amino acids. To obtain support for this L- LIUCINI 10 M FIG. 10. Graphic determination of Ki for L-leucine. possibility, L-glutamine and L-asparagine which Symbols: 0, 7.5 X 10-J M L-valine; A, 5 X 10-5 M lack the negative charge were tested for an effect L-valine; 0, 2 X 10-5 M L-valine. V Micromoles of on the A. conoides system of L-valine uptake. The Ki values for L-glutamine and L-asparagine L-valine uptake per mg (dry weight) per min. -'
0
=
GUPTA AND PRAMER
128
TABLE 5. Competitive inhibition of L-valine uptake by various amino acids and related compoundsa Compound
Inhibition constant
D-Valine ........................ L-Leucine ....................... L-Isoleucine ..................... L-Alanine .......................
4.0 X 105 1.6 X 10-5 2.0 X 105 1.8 X 10-5 L-Phenylalanine ................. 2.2 X 10- 5 2.3 X 10-5 L-Methionine ......... L-Serine ......................... 3.8 X 10-5 L-Cysteine ...................... 4.0 X 10-r Glycine ......................... 2.6 X 10-5 a-Aminoisobutyric acid .......... 4.2 X 10-5 ,3-Alanine ....................... 3.7 X 10-5 .y-Aminobutyric acid ............ 8.0 X 10-5 L-Valine methyl ester ............ Not inhibitory Glycine methyl ester ............ Not inhibitory Isobutylamine ................... Not inhibitory Histamine ....................... Not inhibitory N-monomethyl DL-valine ........ Not inhibitory L-Proline ........................ Not inhibitory as-Ketoisovaleric acid ............ Not inhibitory Butyric acid ..................... Not inhibitory L-Aspartic acid .................. 3.8 X 10-i L-Glutamic acid ................. 5.0 X 10-4 L-Asparagine .................... 2.6 X 10-' L-Glutamine ................... 3.0 X 10-5 L-Histidine ...................... 8.0 X 10-6 L-Lysine ........................ 7.0 X 1a-6 L-Arginine ...................... 2.6 X 10-6
a In the course of these studies the Km for Lvaline uptake varied from 1.5 X 10-5 to 7.5 X
10-' M. were 3 X 10-5 M and 2.6 X 10-5 M, respectively. This is a 10-fold increase in affinity and a change in behavior equal to that of neutral amino acids. This indicated that the removal of the carboxyl group and its associated charge increase affinity of both compounds for the carrier site. The previous results suggested that the site of amino acid transport by A. conoides has less affinity for acidic than for neutral compounds and, therefore, may be anionic itself. If this were true, then basic amino acids may have the greatest affinity for the carrier site, and this would be expressed by reduced Ki values. Tests with Lhistidine, L-arginine, and L-lysine established that this was indeed the case. These compounds had Ki values of 8 X 10-6 M,2.6 X 10-6 M, and 7 X 10-6 M, respectively (Table 5), and these values were at least 1, 0 those of the neutral amino acids and 1f 00 those of the acidic amino acids tested. It is apparent from the foregoing results that all amino acids with unmodified a-carboxyl and
J. BACTERIOL.
a-amino groups compete for the same carrier sites, and affinity for these sites is determined primarily by the ionic species of the molecule. Acidic amino acids had the least affinity (the highest Ki values), basic amino acids had the greatest affinity (the lowest Ki values), and the neutral amino acids were intermediate. The Ki values obtained with many neutral compounds did not differ significantly from the Km value for L-valine. This was interpreted to mean that they were all equally able to combine with the site of amino acid uptake and it follows, therefore, that compounds with Ki values greater than Km had less affinity, and those with Ki values below Km had more affinity than L-valine for the site of amino acid transport in A. conoides. DISCUSSION
The uptake of L-valine by germinated spores of A. conoides was dependent on temperature and had an energy of activation of 16,000 cal/mole and a Qlo value of 2.4. Since free diffusion and other physicochemical phenomena have energy of activation and Qlo values in the order of only 1,000 cal/mole and 1.5, respectively, it appeared that the uptake of L-valine by A. conoides required metabolic involvement and a system that had catalytic or enzyme-like properties (19). Uptake was optimal at pH 5 to 6. Changes in pH can modify valine by influencing ionization of the amino or carboxyl groups of the amino acid. On the other hand, pH may exert an effect by acting on the site of mechanism of valine uptake rather than amino acid itself. Moreover, these two possibilities are not mutually exclusive: pH can simultaneously influence ionization of valine and the cellular site concerned with amino acid uptake. L-Valine was accumulated against a concentration gradient and was not lost from cells by leakage or by exchange. Roess and DeBusk (18) reported a similar lack of leakage or exchange of accumulated argininine even in the presence of energy poisons. Transport was independent of valine incorporation into protein but required energy supplied by metabolic reactions that were inhibited by catalytic amounts of 2,4-dinitrophenol and azide. The foregoing results suggested that L-valine uptake by A. conoides required metabolic energy, i.e., that L-valine uptake was an active transport. The carrier system resembles an enzymatic process in many ways and, like an enzyme reaction, it can be described by Michaelis-Menten kinetics (2, 13, 14, 20). The kinetics of the system were consistent with a mechanism of uptake that depended on a limited number of sites on the cell surface. These loci for valine transport are
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AMINO ACID TRANSPORT BY A. CONOIDES
characterized by a carrier system with an affinity for the amino acid measured as Km value of 1.5 x 10-5 to 7.5 X 10-5 M, and a specificity of particular interest. Cohen and Rickenberg (5) reported that leucine, isoleucine, and valine were accumulated in Escherichia coli by a common mechanism which was not used for the uptake of other amino acids. In every case, only the L-isomer was transported and uptake was inhibited by other compounds that were structurally related. Additional study showed that, for uptake, the amino and carboxyl groups of valine must be unsubstituted, and modification of the carbon chain (substitution of dibutyl for dimethyl groups) resulted in a molecule that was not transported (10). Halvorson and Cohen (7) observed competition between valine and phenylalanine for a common carrier in Saccharomyces cereviseae. Nmethyl-valine and valine amide did not interfere with valine uptake, demonstrating again the essentiality for transport of both the amino and carboxyl groups of the amino acid. With the exception of proline, all the amino acids that were supplied to A. conoides competed with L-valine for a common site on the cell surface. The site has less affinity for acidic amino acids (glutamic and aspartic acids) than for neutral amino acids (leucine, isoleucine, methionine, phenylalanine, alanine, serine, cysteine, glycine, and a-aminoisobutyric acid), and greatest affinity for basic amino acids (histidine, lysine, and arginine). The range of affinities here was in the order of 100, as measured in terms of Ki values for the various compounds in the valine uptake system, and was consistent with a conclusion that, in A. conoides, most if not all amino acids were transported by a common carrier system that possesses an overall negative charge. It is this charge that was responsible for the reduced affinity of the system for acidic amino acids and its greater reactivity with basic amino acids, as compared to neutral compounds. When the negative charge on the side chain of an acidic amino acid was neutralized by converting the carboxyl substituent to an amide, the compound showed enhanced uptake and ability to compete with L-valine for transport to the same extent as other neutral amino acids. The carrier site lacked stereospecificity, and it had no affinity for amino acids unless they retained free a-amino and carboxyl groups. Similar requirements for valine uptake by yeast were noted by Halvorson and Cohen (7), and studies of the filamentous fungus, Botrytis fabae, led Jones (11) to conclude that the organism had a single system by which all amino acids were transported. Figure 12 schematically presents details of a
!I
A
I
4
-x
+
A
,ik
ATP
ADP + Pi A
+
x ADP + Pi
A!
Ix
EXTERIOR OF CELL
ATP :
CELL MEMBRAI ME
AX
INTERIOR OF CELL
FIG. 12. Proposed carrier mechantism for amino acid uptake by A. conoides. A, Amino acid; X, carrier; AX, amino acid-carrier complex; X', inactive carrier.
proposed transport mechanism that is based on models advanced previously by others (8, 9). It is suggested here that the amino acid (A) combines with a carrier (X) to form a complex (AX) at the exterior surface of the cell. The complex moves from the exterior to the interior surface of the cell membrane due to conformational or other changes, and then dissociates, generating inactive carrier (X') and releasing the amino acid (A) to a metabolic pool. This process, and the reactivation of carrier at exterior surface (transformation of X' to X), probably requires energy. In effect, the conversion of adenosine triphosphate to adenosine diphosphate and inorganic phosphate (Pi) provides the force necessary to transport and accumulate Lvaline against a concentration gradient. Since (X') cannot combine with (A) and since reactivation of carrier cannot take place at interior surface, there is no loss of pooled amino acid by exchange or by cell leakage. ACKNOWLEDGMENTS The senior author gratefully acknowledges the assistance of Dexter H. Howard in the preparation of this manuscript.
LITERATURE CITED 1. Ames, G. E. 1964. Uptake of amino acids by Salmonella typhimurium. Arch. Biochem. Biophys. 104:1-18. 2. Benko, P. V., T. C. Wood, and I. H. Segel. 1967. Specificity and regulation of methionine transport in filamentous fungi. Arch. Biochem. Biophys. 122:783-804. 3. Bray, G. A. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1:219-285. 4. Christensen, N. H., and M. J. Liang. 1965. An amino acid transport system of unassigned function in the Ehrlich ascites tumor cells. J. Biol. Chem. 240:3601-3608.
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5. Cohen, G. N., and H. V. Rickenberg. 1956. Concentration specifique reversible des amino acids chez Escherichia coli. Ann. Inst. Pasteur 91:693-720. 6. Dixon, M. 1953. Determination of enzyme inhibitor constant. Biochem. J. 55:170-171. 7. Halvorson, H. O., and G. N. Cohen. 1958. Incorporation des amino acids endogenes dans les proteines de la levure. Ann. Inst. Pasteur 95:73-87. 8. Heinz, E., and P. M. Walsh. 1958. Exchange diffusion, transport and intracellular level of amino acids in Ehrlich carcinoma cells. J. Biol. Chem. 233:1488-1493. 9. Hokin, L. E., and M. R. Hokin. 1963. Phosphatidic acid metabolism and active transport of sodium. Fed. Proc. 22: Part 1, 8-18. 10. Holden, J. T. 1962. The composition of microbial amino acid pools, p. 73-108. In J. H. Holden (ed.), Amino acid pools. Elsevier Publishing Co., New York. 11. Jones, 0. T. G. 1963. The accumulation of amino acids by fungi, with particular reference to the Kant parasitic fungus Botrytisfabae. J. Exp. Bot. 14:399-411. 12. Lineweaver, H., and D. Burk. 1934. The determination of enzyme dissociation constants. J. Amer. Chem. Soc. 56: 658-666. 13. Litwack, G., and D. Pramer. 1956. Absorption of antibiotic by plant cells. III. Kinetics of streptomycin uptake. Arch. Biochem. Biophys. 68:396-402. 14. Mayshak, J., 0. C. Yoder, K. C. Bearman, and D. C. Shelton.
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16. 17.
18. 19.
20. 21.
22.
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1966. Inhibition and transport kinetic studies involving L-leucine, L-valine, and their dipeptides in Leuconostoc mesenteroides. Arch. Biochem. Biophys. 113:189-194. Pramer, D. 1964. Nematode-trapping fungi. Science 144:382388. Pramer, D., and S. Kuyama. 1963. Symposium on biochemical basis of morphogenesis in fungi. I1. Nemin and nematode-trapping fungi. Bacteriol. Rev. 27:282-292. Pramer, D., and N. R. Stoll. 1959. Nemin: a morphogenic substance causing trap formation by predaceous fungi. Science 129:966-967. Roess, W. B., and A. G. DeBusk. 1968. Properties of a basic amino acid permease in Neurospora crassa. J. Gen. Microbiol. 52:421-432. Solomon, A. K. 1952. Permeability of the human erythrocyte to sodium and potassium. J. Gen. Physiol. 36:57-110. Wiley, W. R., and W. H. Matchett. 1966. Tryptophan transport in Neurospora crassa. I. Specificity and kinetics. J. Bacteriol. 92:1698-1705. Yoder, 0. C., K. C. Bearman, and D. C. Shelton. 1967. Membrane specificity of Leuconostoc mesenteroides for the stereoisomeric forms of glycine and valine depeptides. Can. J. Biochem. 45:213-220. Zalokar, M. 1961. Kinetics of amino acid uptake and protein synthesis in Neurospora. Biochim. Biophys. Acta 46:423432.
