JOURNAL oF BACTzRIOLOGY, Mar. 1976, P. 770-775 Copyright C 1976 American Society for Microbiology
Vol. 125, No. 3 Printed in U-SA.
Glutamate Transport in Membrane Vesicles of the Wild-Type Strain and Glutamate-Utilizing Mutants of Escherichia coli SIMONA KAHANE, MENASHE MARCUS, ESTHER METZER, AND YEHESKEL S. HALPERN* Department of Genetics, The Hebrew University, Jerusalem, Israel, and Department of Molecular Biology, Institute of Microbiology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel* Received for publication 28 August 1975
A highly specific energy-dependent glutamate transport system was demonstrated in membrane vesicles of glutamate-utilizing Escherichia coli K-12 mutants. The glutamate transport activity of membranes from the parent strain, unable to grow on glutamate, was very low. With ascorbate-phenazine methosulfate as the electron donor, mutant preparations displayed 17 to 20 times higher activity than did the wild type. However, the affinity of the mutant carrier for L-glutamate remained the same as in the parent strain. Comparative inhibition analysis of glutamate transport in whole cells and membrane vesicles and of in vitro binding of glutamate to a specific periplasmic-binding protein suggests that under certain conditions the latter may be a component of the E. coli K-12 glutamate transport system. source) supplemented with L-methionine (25 j,g/ml); CSlOlMet+, a methionine prototroph derived from CS101; CS7, a glutamate-utilizing mutant of CS101; CS8Met+, a glutamate-utilizing, methionine prototroph, double mutant of CS101. Growth medium. The basal medium of Davis and Mingioli (1) from which citrate was omitted, with 0.5% glycerol as the carbon source, supplemented, where required, with L-methionine (25 ,ug/ml) was used throughout this work. Chemicals. L-Alanine, i-valine, L-aspartic acid, L-glutamic acid, and D-glutamic acid, all A grade, were purchased from Calbiochem, Los Angeles. yMethyl-L-glutamic acid and y-ethyl-L-glutamic acid were products of the California Corporation for Biochemical Research, Los Angeles. y-Benzyl-L-glutamic and a-ketoglutaric acid were obtained from Nutritional Biochemicals Corp., Cleveland, and reduced glutathione was from Sigma Chemical Co., St. Louis. L-[U-'4C]aspartic acid (204 mCi/mmol) and L-[U-'4C]glutamic acid (237 mCi/mmol) were purchased from New England Nuclear Corp., Boston. L[U-_4C]proline (290 mCi/mmol) was from Radiochemical Centre, Amersham, England. Preparation of membrane vesicles. Membrane vesicles were prepared by the method of Kaback (8), with a modification described in the accompanying paper (9). Transport assays. Transport of glutamate by intact cells was determined with nongrowing suspensions in the presence of chloramphenicol as described previously (7). Transport of glutamate, proline, and aspartate by preparations of membrane MATERIALS AND METHODS vesicles was measured by the method of Lombardi Bacterial strains. The following E. coli K-12 and Kaback (11), with minor modifications where strains were used: CS101, a methionine auxotroph, indicated, in the presence of 20 mM NaCl (S. Kaunable to grow on glutamate (as the major carbon hane, M. Marcus, H. Barash, Y. S. Halpern, and H. 770
Wild-type Escherichia coli K-12 cannot grow on glutamate as the sole source of carbon. Glutamate-utilizing mutants of E. coli CS101 were isolated in this laboratory. Comparative studies on the behavior of wild-type and mutant strains disclosed that the ability of the latter to grow on glutamate was due to a severalfold increase in the capacity and rate of glutamate uptake (7, 12). The transport system involved is specific for glutamate, and its formation is controlled by a genetically determined mechanism of repression (9, 12). Recent work with bacterial membrane vesicles demonstrated that these subcellular structures are capable of accumulating amino acids from the medium against a concentration gradient via energy-dependent specific transport systems (11). It seemed to us that a study of the glutamate transport in E. coli K-12 membrane vesicles, uncomplicated by subsequent metabolic events, as to some extent it is in intact cells, might gain for us a better understanding of some of its molecular and regulatory aspects. This paper describes the kinetics of glutamate transport in wild-type and mutant preparations of membrane vesicles and compares the effects of inhibitors on this transport system in intact cells and membrane preparations.
VOL. 125, 1975
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771
R. Kaback, FEBS Lett., in press) (Na+ inhibits proline transport in vesicles of E. coli ML 308-225 [11] but has no effect on the uptake of proline by membrane preparations of E. coli K-12).
RESULTS Comparison of glutamate uptake by membrane preparations from wild-type and glutamate-utilizing mutant cells. n-Lactate-driven transport of i-glutamic acid and L-proline by membrane vesicles of strains CS101Met+ and CS8Met+ is shown in Fig. 1. One can see that the proline transport activities of the two strains were very similar. Very low proline uptake was observed in the absence of the electron donor; addition of 1)-lactate resulted in a 10- to 15-fold stimulation in the rate of uptake. Entirely different results were obtained in regard to glutamate transport. Preparations from the glutamate-utilizing strain CS8Met+ showed high activity of 1)-lactate-stimulated glutamate transport, similar to that of proline. However, membrane preparations of strain CS101Met+, which cannot grow on glutamate as the sole carbon source and shows low activity of glutamate transport by intact cells, exhibited very little 1)-lactate-stimulated glutamate transport. Lineweaver-Burk plots of glutamate uptake by membrane vesicles of wild-type CS101 and glutamate-utilizing mutant CS7 strains, in the presence of ascorbate-phenazine methosulfate as the electron donor, are given in Fig. 2. One can see that, although the maximum rate of uptake was about 17 times higher with the mutant preparation than with wild-type vesicles, the affinity of the carrier for Lrglutamate was the same in both strains (Km = 23 ,LM). These results are similar to those obtained previously with intact cells (7, 12). As shown in Fig. 3, the mutation in strain CS7 was highly specific for glutamate transport. No difference in the kinetic parameters of L-aspartate transport was found between membrane vesicles of CS7 and those of its wild-type parent, CS101. Effect of structural analogues and other amino acids on glutamate transport in membrane vesicles of E. coli K-12 CS7. Transport of glutamate by intact cells of a glutamateutilizingE. coli K-12 mutant CS1 was shown to be inhibited competitively by a number of structural analogues of L-glutamic acid and non-competitively by a number of amino acids (6). Later studies on the effects of inhibitors on in vitro glutamate binding to a periplasmic glutamate-binding protein disclosed a marked similarity between the inhibition patterns of the two systems (1, 2). In the present study we carried out a detailed analysis of the effects of analogues and amino acids on the transport of
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5 5 10 15 10 15 Minutes Minutes FIG. 1. Time curves of L-glutamate and L-proline
uptake by membrane vesicles of wild-type and glutamate-utilizing strains of E. coli K-12. Transport was measured by the method of Lombardi and Kaback (11) with the following modifications. The volume of the uptake mixture was 2 ml, from which 200-pl aliquots were taken at the times indicated, filtered, and washed with 4 ml of 0.1 M LiCl. The glutamate uptake mixture contained L-['4C]glutamic acid (16.7 mCi/mmol, 30 uM) and was supplemented with 20 mM NaCl (Kahane et al., in press). The proline uptake mixture contained L-['4C]proline (71.5 mCi/mmol, 14 pM). D-Lactate served as the source of energy for transport. The reaction was carried out at 30 C and was started by the addition of vesicles (preincubated for 10 min at 30 C) to the uptake mixture. (A) Strain CS101Met+; (B) strain CS8Met+. Proline uptake with no energy source added (0) and with D-lactate (a). Glutamate uptake with no energy source added (A) and with Dlactate (+).
i-glutamate in membrane vesicles of strain CS7. Two of the compounds tested, y-methyl-Lglutamic acid and a-ketoglutaric acid, competitively inhibited gl4tamate uptake (Fig. 4 and 5). The effects of all of the compounds tested are summarized in Table 1. A direct comparison was made between the inhibition patterns of L-glutamate uptake by intact cells and membrane vesicles and of in vitro glutamate binding by binding protein from strain CS7 (Table 2). Among the gamma esters of glutamate tested, only the methyl ester behaved as a competitive inhibitor with similar
772
KAHANE ET AL.
J. BACTERIOL.
-
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0
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0 0 c
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.
L-Glutomate
15 IG m L-Glutomate
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FIG. 2. Kinetics of L-glutamic acid transport in membrane vesicles of wild-type and glutamate-utilizing mutant strains of E. coli K-12. Transport was measured in the presence of ascorbate-phenazine methosulfate as described previously (11). (A) Strain CS1O1; (B) strain CS7.
K, values in all three systems. y-Ethyl-glutamate competitively inhibited transport by intact cells and in vitro binding but was without effect on L-glutamate transport in vesicles. The y-benzyl ester and reduced glutathione were competitive inhibitors of binding but had no effect on transport in either cells or vesicles. DGlutamate was a competitive inhibitor of transport in intact cells but not in vesicles, whereas a-ketoglutarate inhibited both systems in a competitive fashion. L-Aspartate inhibited binding competitively and transport by intact cells non-competitively but had no effect on glutamate transport in membrane preparations. Finally, L-alanine exhibited similar noncompetitive inhibition of transport by intact cells and in vitro binding but did not affect the glutamate transport activity of membrane vesicles. The implication of these results as to the identity of the carrier and the role of the binding protein in glutamate transport is discussed below. DISCUSSION The results described here demonstrate the presence of a highly specific L-glutamate transport system in the cytoplasmic membrane of glutamate-utilizing mutants of E. coli K-12. The activity of this transport system in membranes of wild-type K-12 strains unable to grow on glutamate, although demonstrable, is very low (Fig. 1 and 2). The glutamate transport activity in mutant membrane preparations, in the presence of an artificial electron donor sys-
tem (ascorbate-phenazine methosulfate [11]), is 17 to 20 times higher than that observed in wild-type vesicles (Fig. 2). However, the affinity of the membrane-bound carrier for glutamate is the same in the wild type and the mutant. This situation is similar to that described by us for glutamate uptake by intact cells, except that the glutamate transport activity of intact mutant cells is only four to five times higher than that of the wild type (7, 12). This discrepancy might be indicative of the participation of additional transport systems in glutamate uptake by intact cells. A possible candidate would be the aspartate transport system described by Kay (10). However, the affinity of this system of glutamate (Ki,.ut = 6.4 x 10-4 M) is lower by almost two orders of magnitude than that described in E. coli K-12 CS strains (7, 12). In addition, the aspartate transport system appears to be retained in membrane vesicles (10). Furthermore, glutamate transport in intact cells (5) and in membrane vesicles of E. coli K-12 CS7 (Kahane et al., in press), like that of E. coli B (4, 13), is dependent on sodium ions, which increase the affinity for glutamate but do not affect the maximum velocity of uptake. Kinetic analysis of glutamate uptake by wild-type and mutant cells in the presence of Na+ reveals only one transport component with a Km of 7 to 10 ,uM (5, 7, 12), in spite of the severalfold higher activity of the mutant. An alternative explanation for the greater increase of glutamate transport activity in mu-
GLUTAMATE TRANSPORT IN E. COLI. II.
VOL. 125, 1975 I
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L-Aspartate FIG. 3. Lineweaver-Burk plots of L-aspartic acid transport by membrane vesicles of E. coli K-12 strains CS7 and CS101. Transport was measured in the presence of D-lactate, by the method ofLombardi and Kaback (11), with L-['4C]aspartic acid (204 mCi/mmol). Strain CS101, (+); strain CS7 (0). 6-
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50 25 m L- Glut amate 6-Methyl-L-Glutamic acid,pM FIG. 4. Effect of y-methyl-L-glutamic acid on the kinetics ofL-glutamate uptake by membrane vesicles of E. coli K-12 strain CS7. Membrane vesicles (5 p1) containing 32.5 pg ofprotein were diluted to a final volume of 50 g in the following uptake mixture: potassium phosphate (pH 6.6), 50 mM; magnesium sulfate, 10 mM; lithium-D-lactate, 20 mM; sodium chloride, 20 mM; L-['4C]glutamic acid, 237 mCilmmol; and 'y-methyl-Lglutamic acid at the concentrations shown. Glutamate uptake was measured at 15, 30, 60, and 90 s and plotted, and initial rates were determined from the linear portions of the curves. The data are presented as double-reciprocal plots (A). (B) Ki determination.
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Vol. 125, No. 3 Printed in U-SA.
Glutamate Transport in Membrane Vesicles of the Wild-Type Strain and Glutamate-Utilizing Mutants of Escherichia coli SIMONA KAHANE, MENASHE MARCUS, ESTHER METZER, AND YEHESKEL S. HALPERN* Department of Genetics, The Hebrew University, Jerusalem, Israel, and Department of Molecular Biology, Institute of Microbiology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel* Received for publication 28 August 1975
A highly specific energy-dependent glutamate transport system was demonstrated in membrane vesicles of glutamate-utilizing Escherichia coli K-12 mutants. The glutamate transport activity of membranes from the parent strain, unable to grow on glutamate, was very low. With ascorbate-phenazine methosulfate as the electron donor, mutant preparations displayed 17 to 20 times higher activity than did the wild type. However, the affinity of the mutant carrier for L-glutamate remained the same as in the parent strain. Comparative inhibition analysis of glutamate transport in whole cells and membrane vesicles and of in vitro binding of glutamate to a specific periplasmic-binding protein suggests that under certain conditions the latter may be a component of the E. coli K-12 glutamate transport system. source) supplemented with L-methionine (25 j,g/ml); CSlOlMet+, a methionine prototroph derived from CS101; CS7, a glutamate-utilizing mutant of CS101; CS8Met+, a glutamate-utilizing, methionine prototroph, double mutant of CS101. Growth medium. The basal medium of Davis and Mingioli (1) from which citrate was omitted, with 0.5% glycerol as the carbon source, supplemented, where required, with L-methionine (25 ,ug/ml) was used throughout this work. Chemicals. L-Alanine, i-valine, L-aspartic acid, L-glutamic acid, and D-glutamic acid, all A grade, were purchased from Calbiochem, Los Angeles. yMethyl-L-glutamic acid and y-ethyl-L-glutamic acid were products of the California Corporation for Biochemical Research, Los Angeles. y-Benzyl-L-glutamic and a-ketoglutaric acid were obtained from Nutritional Biochemicals Corp., Cleveland, and reduced glutathione was from Sigma Chemical Co., St. Louis. L-[U-'4C]aspartic acid (204 mCi/mmol) and L-[U-'4C]glutamic acid (237 mCi/mmol) were purchased from New England Nuclear Corp., Boston. L[U-_4C]proline (290 mCi/mmol) was from Radiochemical Centre, Amersham, England. Preparation of membrane vesicles. Membrane vesicles were prepared by the method of Kaback (8), with a modification described in the accompanying paper (9). Transport assays. Transport of glutamate by intact cells was determined with nongrowing suspensions in the presence of chloramphenicol as described previously (7). Transport of glutamate, proline, and aspartate by preparations of membrane MATERIALS AND METHODS vesicles was measured by the method of Lombardi Bacterial strains. The following E. coli K-12 and Kaback (11), with minor modifications where strains were used: CS101, a methionine auxotroph, indicated, in the presence of 20 mM NaCl (S. Kaunable to grow on glutamate (as the major carbon hane, M. Marcus, H. Barash, Y. S. Halpern, and H. 770
Wild-type Escherichia coli K-12 cannot grow on glutamate as the sole source of carbon. Glutamate-utilizing mutants of E. coli CS101 were isolated in this laboratory. Comparative studies on the behavior of wild-type and mutant strains disclosed that the ability of the latter to grow on glutamate was due to a severalfold increase in the capacity and rate of glutamate uptake (7, 12). The transport system involved is specific for glutamate, and its formation is controlled by a genetically determined mechanism of repression (9, 12). Recent work with bacterial membrane vesicles demonstrated that these subcellular structures are capable of accumulating amino acids from the medium against a concentration gradient via energy-dependent specific transport systems (11). It seemed to us that a study of the glutamate transport in E. coli K-12 membrane vesicles, uncomplicated by subsequent metabolic events, as to some extent it is in intact cells, might gain for us a better understanding of some of its molecular and regulatory aspects. This paper describes the kinetics of glutamate transport in wild-type and mutant preparations of membrane vesicles and compares the effects of inhibitors on this transport system in intact cells and membrane preparations.
VOL. 125, 1975
GLUTAMATE TRANSPORT IN E. COLI. II.
771
R. Kaback, FEBS Lett., in press) (Na+ inhibits proline transport in vesicles of E. coli ML 308-225 [11] but has no effect on the uptake of proline by membrane preparations of E. coli K-12).
RESULTS Comparison of glutamate uptake by membrane preparations from wild-type and glutamate-utilizing mutant cells. n-Lactate-driven transport of i-glutamic acid and L-proline by membrane vesicles of strains CS101Met+ and CS8Met+ is shown in Fig. 1. One can see that the proline transport activities of the two strains were very similar. Very low proline uptake was observed in the absence of the electron donor; addition of 1)-lactate resulted in a 10- to 15-fold stimulation in the rate of uptake. Entirely different results were obtained in regard to glutamate transport. Preparations from the glutamate-utilizing strain CS8Met+ showed high activity of 1)-lactate-stimulated glutamate transport, similar to that of proline. However, membrane preparations of strain CS101Met+, which cannot grow on glutamate as the sole carbon source and shows low activity of glutamate transport by intact cells, exhibited very little 1)-lactate-stimulated glutamate transport. Lineweaver-Burk plots of glutamate uptake by membrane vesicles of wild-type CS101 and glutamate-utilizing mutant CS7 strains, in the presence of ascorbate-phenazine methosulfate as the electron donor, are given in Fig. 2. One can see that, although the maximum rate of uptake was about 17 times higher with the mutant preparation than with wild-type vesicles, the affinity of the carrier for Lrglutamate was the same in both strains (Km = 23 ,LM). These results are similar to those obtained previously with intact cells (7, 12). As shown in Fig. 3, the mutation in strain CS7 was highly specific for glutamate transport. No difference in the kinetic parameters of L-aspartate transport was found between membrane vesicles of CS7 and those of its wild-type parent, CS101. Effect of structural analogues and other amino acids on glutamate transport in membrane vesicles of E. coli K-12 CS7. Transport of glutamate by intact cells of a glutamateutilizingE. coli K-12 mutant CS1 was shown to be inhibited competitively by a number of structural analogues of L-glutamic acid and non-competitively by a number of amino acids (6). Later studies on the effects of inhibitors on in vitro glutamate binding to a periplasmic glutamate-binding protein disclosed a marked similarity between the inhibition patterns of the two systems (1, 2). In the present study we carried out a detailed analysis of the effects of analogues and amino acids on the transport of
c
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5 5 10 15 10 15 Minutes Minutes FIG. 1. Time curves of L-glutamate and L-proline
uptake by membrane vesicles of wild-type and glutamate-utilizing strains of E. coli K-12. Transport was measured by the method of Lombardi and Kaback (11) with the following modifications. The volume of the uptake mixture was 2 ml, from which 200-pl aliquots were taken at the times indicated, filtered, and washed with 4 ml of 0.1 M LiCl. The glutamate uptake mixture contained L-['4C]glutamic acid (16.7 mCi/mmol, 30 uM) and was supplemented with 20 mM NaCl (Kahane et al., in press). The proline uptake mixture contained L-['4C]proline (71.5 mCi/mmol, 14 pM). D-Lactate served as the source of energy for transport. The reaction was carried out at 30 C and was started by the addition of vesicles (preincubated for 10 min at 30 C) to the uptake mixture. (A) Strain CS101Met+; (B) strain CS8Met+. Proline uptake with no energy source added (0) and with D-lactate (a). Glutamate uptake with no energy source added (A) and with Dlactate (+).
i-glutamate in membrane vesicles of strain CS7. Two of the compounds tested, y-methyl-Lglutamic acid and a-ketoglutaric acid, competitively inhibited gl4tamate uptake (Fig. 4 and 5). The effects of all of the compounds tested are summarized in Table 1. A direct comparison was made between the inhibition patterns of L-glutamate uptake by intact cells and membrane vesicles and of in vitro glutamate binding by binding protein from strain CS7 (Table 2). Among the gamma esters of glutamate tested, only the methyl ester behaved as a competitive inhibitor with similar
772
KAHANE ET AL.
J. BACTERIOL.
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.E 0
0
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0
0 0 c
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a
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L-Glutomate
15 IG m L-Glutomate
xIO PM i
FIG. 2. Kinetics of L-glutamic acid transport in membrane vesicles of wild-type and glutamate-utilizing mutant strains of E. coli K-12. Transport was measured in the presence of ascorbate-phenazine methosulfate as described previously (11). (A) Strain CS1O1; (B) strain CS7.
K, values in all three systems. y-Ethyl-glutamate competitively inhibited transport by intact cells and in vitro binding but was without effect on L-glutamate transport in vesicles. The y-benzyl ester and reduced glutathione were competitive inhibitors of binding but had no effect on transport in either cells or vesicles. DGlutamate was a competitive inhibitor of transport in intact cells but not in vesicles, whereas a-ketoglutarate inhibited both systems in a competitive fashion. L-Aspartate inhibited binding competitively and transport by intact cells non-competitively but had no effect on glutamate transport in membrane preparations. Finally, L-alanine exhibited similar noncompetitive inhibition of transport by intact cells and in vitro binding but did not affect the glutamate transport activity of membrane vesicles. The implication of these results as to the identity of the carrier and the role of the binding protein in glutamate transport is discussed below. DISCUSSION The results described here demonstrate the presence of a highly specific L-glutamate transport system in the cytoplasmic membrane of glutamate-utilizing mutants of E. coli K-12. The activity of this transport system in membranes of wild-type K-12 strains unable to grow on glutamate, although demonstrable, is very low (Fig. 1 and 2). The glutamate transport activity in mutant membrane preparations, in the presence of an artificial electron donor sys-
tem (ascorbate-phenazine methosulfate [11]), is 17 to 20 times higher than that observed in wild-type vesicles (Fig. 2). However, the affinity of the membrane-bound carrier for glutamate is the same in the wild type and the mutant. This situation is similar to that described by us for glutamate uptake by intact cells, except that the glutamate transport activity of intact mutant cells is only four to five times higher than that of the wild type (7, 12). This discrepancy might be indicative of the participation of additional transport systems in glutamate uptake by intact cells. A possible candidate would be the aspartate transport system described by Kay (10). However, the affinity of this system of glutamate (Ki,.ut = 6.4 x 10-4 M) is lower by almost two orders of magnitude than that described in E. coli K-12 CS strains (7, 12). In addition, the aspartate transport system appears to be retained in membrane vesicles (10). Furthermore, glutamate transport in intact cells (5) and in membrane vesicles of E. coli K-12 CS7 (Kahane et al., in press), like that of E. coli B (4, 13), is dependent on sodium ions, which increase the affinity for glutamate but do not affect the maximum velocity of uptake. Kinetic analysis of glutamate uptake by wild-type and mutant cells in the presence of Na+ reveals only one transport component with a Km of 7 to 10 ,uM (5, 7, 12), in spite of the severalfold higher activity of the mutant. An alternative explanation for the greater increase of glutamate transport activity in mu-
GLUTAMATE TRANSPORT IN E. COLI. II.
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L-Aspartate FIG. 3. Lineweaver-Burk plots of L-aspartic acid transport by membrane vesicles of E. coli K-12 strains CS7 and CS101. Transport was measured in the presence of D-lactate, by the method ofLombardi and Kaback (11), with L-['4C]aspartic acid (204 mCi/mmol). Strain CS101, (+); strain CS7 (0). 6-
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50 25 m L- Glut amate 6-Methyl-L-Glutamic acid,pM FIG. 4. Effect of y-methyl-L-glutamic acid on the kinetics ofL-glutamate uptake by membrane vesicles of E. coli K-12 strain CS7. Membrane vesicles (5 p1) containing 32.5 pg ofprotein were diluted to a final volume of 50 g in the following uptake mixture: potassium phosphate (pH 6.6), 50 mM; magnesium sulfate, 10 mM; lithium-D-lactate, 20 mM; sodium chloride, 20 mM; L-['4C]glutamic acid, 237 mCilmmol; and 'y-methyl-Lglutamic acid at the concentrations shown. Glutamate uptake was measured at 15, 30, 60, and 90 s and plotted, and initial rates were determined from the linear portions of the curves. The data are presented as double-reciprocal plots (A). (B) Ki determination.
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