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4-Amino-5-imidazolecarboxamide. The relation of its metabolism to citric acid cycle
4-Amino-Hmidazolecarboxamide. The Relation of Its Metabolism to Citric Acid Cycle’ M. G. Sevag and R. C. Stewart From the Department of Microbiology, School of Medicine, Pennsylvania, Philadelphia, Pennsylvania Received
July
Universily
of
2, 1952
INTRODUCTION
Fox (1) made the important discovery that. a non-acetylatable, diazotizable amine accumulates during the growth of Escherichia coli in salt-glucose medium in the presence of growth-inhibitory concentrations of a sulfonamide. Stetten and Fox (2) isolated and determined certain properties of this amine. It was subsequently characterized as 4-amino5-imidazolecarboxamide2 by Shive et al. (3). Miller, Gurin, and Wilson (4) reported that adenine and guanine of the nucleic acids and adenine of nucleotides may be derived in large measure from isotopically labeled 4-amino&imidazolecarboxamide when injected into rats. Similarly, Conzelman et al. (5) reported that labeled amine injected into tumor-bearing mice yielded ribonucleic acid (RNA) and desoxyribonucleic acid (DNA) containing most of the activity. From results obtained with pigeon liver experiments, Schulman and Buchanan (6) suggested that this substance, in the form of its ribotide, may be a key intermediate in the synthesis of purines. Williams (7), using extracts and suspensions of intact Saccharomyces cerevisiae, reported that labeled carboxamide is converted to adenine and guanine. These observations indicate that 4-amino-5-imidazolecarbox?mide is a purine intermediary, or convertible into an intermediary, and may exercise an important role in the synthesis of purines. In the preceding communication (8) it was reported that the accumulation of this amine 1 This work was aided by a contract between the Office of Naval Research, Department of the Navy, and the University of Pennsylvania. 2 For brevity, the terms amine or carboxamide are used in this paper.
14
4-AMINO-5-IMIDAZOLECARBOXAMIDE.
15
II
during the growth of a wild strain of E: coli B in casein hydrolyzate medium is dependent upon the presence of a carbohydrate such as glucose or L-arabinose, etc. Data presented here suggest that the metabolism of purines and the accumulation of this amine may in somemanner be linked to the functioning of the Krebs’ citric acid cycle. METHODS The growth medium used in this study and the method of determining are the same as those used in the preceding communication (8).
the amine
'7-
0
10
2” TIME
30 40 IN HOURS
50
60
FIG. 1. The effect of aeration on the accumulation of 4-amino-5-imidazolecarbosamide in E. coli B in the presence of 5 pg. sulfathiazole/ml.
RESULTS
The data presented in Fig. 1 show that intense aeration (magnetic stirring) at the start of the growth period of the systems containing 5 pg. sulfathiazole/ml. almost completely inhibits the accumulation of the amine. In simultaneous experiments, another identical growth system was allowed to incubate without aeration for 25 hr. It was then divided into two equal parts: one was aerated magnetically during incubation, and the other allowed to incubate without aeration or stirring. It can be seen that at the end of the 25-hr. growth period about 3 rg./ml: of amine had accumulated. This amount increased to 4 pg./ml. in the aerated system during an incubation period of 60 hr. However, 14.25 H. of amine/ml. accumulated in the nonaerated system during the same period.
16
Inhibition
SEVAG
ZZZ I w/ml.
Additions
Growth turbidity 17 hr. ti.5 hr _-
Acetate
.Malate
.Malonate
.Succinate
45 --
65
115 5.0 115 5.2 116 5.2 119 5.2 123 5.2 - -. __
115 100 74 67 77 --
115 100 90 90 77
133 143 137
4.8 2.8 1.8 -. __
2 2 2
--
123 122 135 -
5.0 5.2 5.2 5.2 5.2 -I_ 4.7 2.8 1.8 --
115 100 52 43 40
--
114 111 115 111 116 -
60 55 60 --
121 124 121
2.4 0 0
2.7 0 0 --
5 2 2
--
100 117 118 -
190 204 140
2.6 0 0
--
113 128 110 -
2.4 0 0 -__
142 162 146
3.8 0 0
--
104 132 125 -
--
107 130 95 -
.or-Ketoglutarate
1000 3000 5000 1000 3000 5000 1000 3000 5000 1000 3000 5000
loo0 3000 5ooo --
124
_--
.-
.-
_-
-. __
_.-
-. -
_.-
152 1.9 169 0 125 0 .- - -_I_ 146 156 4.3 140 165 4.2 116 185 3.1 -_-
117 105 112
147 160 170
4.3 3.0 0
65 hr
-
17
hr.
41
hr.
65 hr.
0
9.4
6.8 10.6 16.5 16.3 15.6
5.8 8.2 13.5 12.7 14.4
5.4 9.2 13.7 13.7 15.6
61 82 88
0 0 0
15.6 12.3 13.4
21.2 16.5 17.0
64 95 20 __-
62 105 105
0 0 0
13.5 9.4 0
18.2 14.1 5.8
12 124 98
59 130 120 --
115 165 147
0 4.0 0 __
10.1 4.7 0
4.0 0 0 ----
3 51 112
41 95 132 ---
115 114 147
0 4.9 0.2
10.8 7.6 1.0
2.0 0 0 ____ 4.7 4.0 3.4 -__
10 113 82
49 130 95 -47 32 64 ___ 41 41 85
90 147 113
0 1.1 0
11.5 1.0 0
14.7 1.9 0
66 89 115
0 0 0
10.4 8.4 8.0
15.,6 14.5 10.7
68 82 120
0 0 4.0
10.8 13.6 11.4
15.8 16.7 12.6
4 8 10
4.3 8 3.4 8 34 0 _-___ (1This value ranged from 3 to 5 fig. in numerous 1ooo 13006 ‘i 5000
Amine, ag.lml.
-- -
0
1000 3000 5000
Fumarate
17 hr. 4 1 hr. .- --
4.7 --
5.0 0.5 0.2 0.1 0.02 1
.-I
-~
I
Growth turbidity
4.7
111 --
Pyruvate
Amine, Pg./ml.
by the Acids of
5 fig. sulfathiazole/ml.
I
65 hr. ~__
-
PABA
I
Control
17 hr
--
None
STEWART
TABLE I of 4-AminoJ-imidazolecarboxamide Krebs’ Cycle
of the Accumulation
Medium 1% casein hydrol. yzate + o.12s~o glucose
AND
-
--
--
--
_-
-
experiments.
_-
.-
.-
.-
-
15.3
12.2 5.4 0 13.8 8.2 1.0
4-AMINO-&IMIDAZOLECARBOXAMIDE.
II
17
The data presented in Table I show that five concentrations of paminobenzoic acid (PABA), ranging from 5.0 to 0.02 pg./ml., failed to prevent the accumulation of the amine in normal growth systems. In systems containing 5 pg./ml. of sulfathiazole, 5.0 and 0.5 pg. of PABA/ml. antagonized the growth inhibition without completely inhibiting or blocking the accumulation of the amine. With these two concentrations of PABA the amount of amine is approximately the same as that found in sulfathiazole-free systems. The possibility is that PABA blocked only the accumulation of that fraction of the total amount of amine which is related to the action of sulfathiazole. In the other three lower ccncentrations of PABA, growth inhibition is partially antagonized. However, the accumulation of the amine is not affected; the amounts found are of about the same magnitude as those in the systems free from PABA. Data not reported here likewise show that from 3.0 to 0.03 pg./ml. of citrovorum factor (kindly supplied by Dr. J. M. Buchanan of the Department of Physiological Chemistry) failed to exercise any effect on the accumulation of the amine in all systems. It also failed to exercise any effect on the degree of inhibition by sulfathiazole. Some of the mono- and dicarboxylic acids related to the citric acid cycle exercise remarkable effects on the accumulation of the amine and the inhibition of growth. Of these acids, fumaric failed to exercise any effect on these systems. Pyruvate reduced the amount of the amine accumulated in the sulfathiazole-free systems by 60% but failed to exercise a similar effect on sulfathiazole-containing systems. Acetate inhibited the accumulation of the amine in sulfathiazole-free and sulfathiazole-containing systems, without antagonizing the inhibition of growth. a-Ketoglutarate inhibited the accumulation in control systems and antagonized the inhibition of growth without, however, influencing the accumulation of the amine in these sulfathiazole-inhibited systems. Malate, malonate, and succinate were the most effective substances tested. They inhibited the accumulation of the amine in all systems and completely antagonized the inhibition of growth by sulfathiazole. However, although the growth inhibition is antagonized, a parallel decrease in the accumulation of amine is not evident in sulfathiazole-containing systems. Some of the other components of the Krebs’ cycle (e. g., citric acid) exercise similar effects and will be reported in a later communication.
18
SEVAG
AND
STEWART
DIscussroh-
The data presented here would appear to show clearly that certain steps in carbohydrate metabolism are intimately linked with the accumulation of 4-aminod-imidazolecarboxamide, or with the metabolism of purines. Inhibition of the accumulation of amine by aeration may be the result of the intensification of aerobic metabolism of glucose. Inhibition by sulfathiazole of the dismutation of pyruvate (9, lo), aerobic oxidation of lactic acid (9), the citric acid cycle (11, 12), and also the fact that sulfathiazole causes a several-fold increment in amine accumulation, suggest the possibility that an impairment or interference with the citric acid cycle is responsible for the accumulation of the carboxamide. Aeration, favoring the optimal functioning of the citric acid cycle, inhibits the accumulation of the amine. On this basis, it would appear that sulfathiazole bacteriostasis may involve a depression of the aerobic and a stimulation of the anaerobic metabolism of glucose, causing, under the latter condition, the increase in the amount of the amine accumulated. This interpretation is in agreement with the fact that in sulfathiaiole-free anaerobic growth systems, from 35 to 40% more amine is accumulated than in aerobic systems. The indicated interference with the citric acid cycle may mean that certain critical substances which are required to complete the purine molecule, or to provide the required energy, are not being produced in adequate amounts. This suggested the possibility that by providing the growth systems exogenously with the components of the citric acid cycle, the postulated impairment of the citric acid cycle could be remedied. As shown in Table I, certain components of the citric acid cycle: acetate, malate, sue&ate, etc., and malonate abolished the accumulation of the amine and antagonized the inhibition of growth by sulfathiazole. In what manner these substances exercise the foregoing effects is a question for detailed study in the future. It may be mentioned here that the effects exercised by these acids do not appear to be by displacement of sulfathiazole, but rather, by functioning as metabolites, the synthesis of which were blocked. This is demonstrated by the fact that these components of the citric acid cycle also block the accumulation of the carboxamide in systems which are free from sulfathiazole. Pertinent also is the fact that in preliminary experiments, these components of the citric acid cycle failed to antagonize the inhibition by 5 pg./ml. of sulfathiazole in growth systems free from carbohydrates. Here, apparently, the citric acid cycle is probably operating at a reduced rate and the
4-AMIh’O-j-IMIDAZOLECARBOXAMIDE.
II
19
inhibition of growth must involve enzyme systems ot.her than those of the citric acid cycle and may be concerned with the amino acid metabolism . SUM~~ARY
AND CONCLUSION
Factors controlling the prevention of t.he accumulation of 4-amino-5imidazolecarboxamide during the growth of Escherichia wli R in 1% casein hydrolyzate medium containing 0.125% glucose were studied. p-hminobenzoic acid (PAnil) at cert,ain concentrations antagonized the inhibition of growth by sulfathiazole but failed to prevent the accumulation of the carboxamide in bot.h control and sulfathiazolecontaining systems. Citrovorum factor failed both to prevent t.he a+ cumulation of the amine and to antagonize the inhibition of growth. These facts may permit the conclusion t,hat PAPA antagonism to sulfathiazole inhibition of growt,h, and the metabolism of the carboxamide as a purine intermediary are independent processes, or the increase in amount of the accumulated carboxamide in t.hc presence of sulfathiazole may be due primarily to the effect of sulfathiazole on the citric acid cycle, and t.o inhibition of purine synthesis only secondarily to this primary inhibition. Aeration, both at the start. and during the incubation of the growth systems, prevents the accumulation of t.he carboxamide. This can be interpreted to indicate that the accumulation of the carboxamide is related to an impairment of the aerobic and the promotion of anaerobic metabolism. The increment in the amount of the carboxamide accumulated during sulfathiazole bacteriostasis may likewise indicate that sulfathiazole suppresses the aerobic and promotes the anaerobic metabolism of the system. Comparable to the effect obtained during aeration, the addition to the growth systems of mono- and dicarboxylic acids related t.o the citric arid cycle inhibits the accumulation of the carboxamide. These acids also antagonize t,he inhibition of growth by sulfathiazolc.
1. Fox,
C. L., JR., hoc. Sot. Ezpll. Riol. .Ued. 61, 102. (1942). M. It., ASD Fox, C. I,., *JR., J. Hiol. Chrm. 161.333 (1946). 3. SIIIVE, W., ACKER~~IASS, W. W., CORDOK, >I., GETAESDANER, M. E., ANI) E:AKIX, 11. E., J. Aw. Chenr. Sot. 69. 725 (1947). 4. MILLER, C. S., GURIS, S., ASI) WIISO~, n. W., Science 111, 654 (1950). 5. CONZELMAX, G. M.,JR.,MAsDEL. H. G., ASD SMITH, I'. K., Federation Proc. 11, 199 (1962). 2. STETITN,
20
SEVAG
AND
STEWART
6. SCHULI~AN, M. P., AND BUCHANAN, J. M., Federation Proc. J. Biol. Chem. 196, 513 (1952). 7. WILLIAMS, W. J., Federation Proc. 10, 270 (1951). 8. STEWART, R. C., AND SEVAG, M. G., Arch. Biochem. Biophys. 9. Fox, C. L., JR., J. Bact. 43, 68 (1942). 10. SEVAG, M. G., SHELBURNE, M., AND MIJDD, S., J. Bact. 49, 65 11. STEERS, E., AND SEVAG, M. G., Arch. Biochem. 24, 129 (1949). 12. SEVAG, M. G., GOTS, J. S., AND STEERS, E., in The Enzymes, p. 115. Academic Press, New York, 1950.
10, 244 (1951);
41, 9 (1952). (1945). Vol. I, Part
I,
July
Universily
of
2, 1952
INTRODUCTION
Fox (1) made the important discovery that. a non-acetylatable, diazotizable amine accumulates during the growth of Escherichia coli in salt-glucose medium in the presence of growth-inhibitory concentrations of a sulfonamide. Stetten and Fox (2) isolated and determined certain properties of this amine. It was subsequently characterized as 4-amino5-imidazolecarboxamide2 by Shive et al. (3). Miller, Gurin, and Wilson (4) reported that adenine and guanine of the nucleic acids and adenine of nucleotides may be derived in large measure from isotopically labeled 4-amino&imidazolecarboxamide when injected into rats. Similarly, Conzelman et al. (5) reported that labeled amine injected into tumor-bearing mice yielded ribonucleic acid (RNA) and desoxyribonucleic acid (DNA) containing most of the activity. From results obtained with pigeon liver experiments, Schulman and Buchanan (6) suggested that this substance, in the form of its ribotide, may be a key intermediate in the synthesis of purines. Williams (7), using extracts and suspensions of intact Saccharomyces cerevisiae, reported that labeled carboxamide is converted to adenine and guanine. These observations indicate that 4-amino-5-imidazolecarbox?mide is a purine intermediary, or convertible into an intermediary, and may exercise an important role in the synthesis of purines. In the preceding communication (8) it was reported that the accumulation of this amine 1 This work was aided by a contract between the Office of Naval Research, Department of the Navy, and the University of Pennsylvania. 2 For brevity, the terms amine or carboxamide are used in this paper.
14
4-AMINO-5-IMIDAZOLECARBOXAMIDE.
15
II
during the growth of a wild strain of E: coli B in casein hydrolyzate medium is dependent upon the presence of a carbohydrate such as glucose or L-arabinose, etc. Data presented here suggest that the metabolism of purines and the accumulation of this amine may in somemanner be linked to the functioning of the Krebs’ citric acid cycle. METHODS The growth medium used in this study and the method of determining are the same as those used in the preceding communication (8).
the amine
'7-
0
10
2” TIME
30 40 IN HOURS
50
60
FIG. 1. The effect of aeration on the accumulation of 4-amino-5-imidazolecarbosamide in E. coli B in the presence of 5 pg. sulfathiazole/ml.
RESULTS
The data presented in Fig. 1 show that intense aeration (magnetic stirring) at the start of the growth period of the systems containing 5 pg. sulfathiazole/ml. almost completely inhibits the accumulation of the amine. In simultaneous experiments, another identical growth system was allowed to incubate without aeration for 25 hr. It was then divided into two equal parts: one was aerated magnetically during incubation, and the other allowed to incubate without aeration or stirring. It can be seen that at the end of the 25-hr. growth period about 3 rg./ml: of amine had accumulated. This amount increased to 4 pg./ml. in the aerated system during an incubation period of 60 hr. However, 14.25 H. of amine/ml. accumulated in the nonaerated system during the same period.
16
Inhibition
SEVAG
ZZZ I w/ml.
Additions
Growth turbidity 17 hr. ti.5 hr _-
Acetate
.Malate
.Malonate
.Succinate
45 --
65
115 5.0 115 5.2 116 5.2 119 5.2 123 5.2 - -. __
115 100 74 67 77 --
115 100 90 90 77
133 143 137
4.8 2.8 1.8 -. __
2 2 2
--
123 122 135 -
5.0 5.2 5.2 5.2 5.2 -I_ 4.7 2.8 1.8 --
115 100 52 43 40
--
114 111 115 111 116 -
60 55 60 --
121 124 121
2.4 0 0
2.7 0 0 --
5 2 2
--
100 117 118 -
190 204 140
2.6 0 0
--
113 128 110 -
2.4 0 0 -__
142 162 146
3.8 0 0
--
104 132 125 -
--
107 130 95 -
.or-Ketoglutarate
1000 3000 5000 1000 3000 5000 1000 3000 5000 1000 3000 5000
loo0 3000 5ooo --
124
_--
.-
.-
_-
-. __
_.-
-. -
_.-
152 1.9 169 0 125 0 .- - -_I_ 146 156 4.3 140 165 4.2 116 185 3.1 -_-
117 105 112
147 160 170
4.3 3.0 0
65 hr
-
17
hr.
41
hr.
65 hr.
0
9.4
6.8 10.6 16.5 16.3 15.6
5.8 8.2 13.5 12.7 14.4
5.4 9.2 13.7 13.7 15.6
61 82 88
0 0 0
15.6 12.3 13.4
21.2 16.5 17.0
64 95 20 __-
62 105 105
0 0 0
13.5 9.4 0
18.2 14.1 5.8
12 124 98
59 130 120 --
115 165 147
0 4.0 0 __
10.1 4.7 0
4.0 0 0 ----
3 51 112
41 95 132 ---
115 114 147
0 4.9 0.2
10.8 7.6 1.0
2.0 0 0 ____ 4.7 4.0 3.4 -__
10 113 82
49 130 95 -47 32 64 ___ 41 41 85
90 147 113
0 1.1 0
11.5 1.0 0
14.7 1.9 0
66 89 115
0 0 0
10.4 8.4 8.0
15.,6 14.5 10.7
68 82 120
0 0 4.0
10.8 13.6 11.4
15.8 16.7 12.6
4 8 10
4.3 8 3.4 8 34 0 _-___ (1This value ranged from 3 to 5 fig. in numerous 1ooo 13006 ‘i 5000
Amine, ag.lml.
-- -
0
1000 3000 5000
Fumarate
17 hr. 4 1 hr. .- --
4.7 --
5.0 0.5 0.2 0.1 0.02 1
.-I
-~
I
Growth turbidity
4.7
111 --
Pyruvate
Amine, Pg./ml.
by the Acids of
5 fig. sulfathiazole/ml.
I
65 hr. ~__
-
PABA
I
Control
17 hr
--
None
STEWART
TABLE I of 4-AminoJ-imidazolecarboxamide Krebs’ Cycle
of the Accumulation
Medium 1% casein hydrol. yzate + o.12s~o glucose
AND
-
--
--
--
_-
-
experiments.
_-
.-
.-
.-
-
15.3
12.2 5.4 0 13.8 8.2 1.0
4-AMINO-&IMIDAZOLECARBOXAMIDE.
II
17
The data presented in Table I show that five concentrations of paminobenzoic acid (PABA), ranging from 5.0 to 0.02 pg./ml., failed to prevent the accumulation of the amine in normal growth systems. In systems containing 5 pg./ml. of sulfathiazole, 5.0 and 0.5 pg. of PABA/ml. antagonized the growth inhibition without completely inhibiting or blocking the accumulation of the amine. With these two concentrations of PABA the amount of amine is approximately the same as that found in sulfathiazole-free systems. The possibility is that PABA blocked only the accumulation of that fraction of the total amount of amine which is related to the action of sulfathiazole. In the other three lower ccncentrations of PABA, growth inhibition is partially antagonized. However, the accumulation of the amine is not affected; the amounts found are of about the same magnitude as those in the systems free from PABA. Data not reported here likewise show that from 3.0 to 0.03 pg./ml. of citrovorum factor (kindly supplied by Dr. J. M. Buchanan of the Department of Physiological Chemistry) failed to exercise any effect on the accumulation of the amine in all systems. It also failed to exercise any effect on the degree of inhibition by sulfathiazole. Some of the mono- and dicarboxylic acids related to the citric acid cycle exercise remarkable effects on the accumulation of the amine and the inhibition of growth. Of these acids, fumaric failed to exercise any effect on these systems. Pyruvate reduced the amount of the amine accumulated in the sulfathiazole-free systems by 60% but failed to exercise a similar effect on sulfathiazole-containing systems. Acetate inhibited the accumulation of the amine in sulfathiazole-free and sulfathiazole-containing systems, without antagonizing the inhibition of growth. a-Ketoglutarate inhibited the accumulation in control systems and antagonized the inhibition of growth without, however, influencing the accumulation of the amine in these sulfathiazole-inhibited systems. Malate, malonate, and succinate were the most effective substances tested. They inhibited the accumulation of the amine in all systems and completely antagonized the inhibition of growth by sulfathiazole. However, although the growth inhibition is antagonized, a parallel decrease in the accumulation of amine is not evident in sulfathiazole-containing systems. Some of the other components of the Krebs’ cycle (e. g., citric acid) exercise similar effects and will be reported in a later communication.
18
SEVAG
AND
STEWART
DIscussroh-
The data presented here would appear to show clearly that certain steps in carbohydrate metabolism are intimately linked with the accumulation of 4-aminod-imidazolecarboxamide, or with the metabolism of purines. Inhibition of the accumulation of amine by aeration may be the result of the intensification of aerobic metabolism of glucose. Inhibition by sulfathiazole of the dismutation of pyruvate (9, lo), aerobic oxidation of lactic acid (9), the citric acid cycle (11, 12), and also the fact that sulfathiazole causes a several-fold increment in amine accumulation, suggest the possibility that an impairment or interference with the citric acid cycle is responsible for the accumulation of the carboxamide. Aeration, favoring the optimal functioning of the citric acid cycle, inhibits the accumulation of the amine. On this basis, it would appear that sulfathiazole bacteriostasis may involve a depression of the aerobic and a stimulation of the anaerobic metabolism of glucose, causing, under the latter condition, the increase in the amount of the amine accumulated. This interpretation is in agreement with the fact that in sulfathiaiole-free anaerobic growth systems, from 35 to 40% more amine is accumulated than in aerobic systems. The indicated interference with the citric acid cycle may mean that certain critical substances which are required to complete the purine molecule, or to provide the required energy, are not being produced in adequate amounts. This suggested the possibility that by providing the growth systems exogenously with the components of the citric acid cycle, the postulated impairment of the citric acid cycle could be remedied. As shown in Table I, certain components of the citric acid cycle: acetate, malate, sue&ate, etc., and malonate abolished the accumulation of the amine and antagonized the inhibition of growth by sulfathiazole. In what manner these substances exercise the foregoing effects is a question for detailed study in the future. It may be mentioned here that the effects exercised by these acids do not appear to be by displacement of sulfathiazole, but rather, by functioning as metabolites, the synthesis of which were blocked. This is demonstrated by the fact that these components of the citric acid cycle also block the accumulation of the carboxamide in systems which are free from sulfathiazole. Pertinent also is the fact that in preliminary experiments, these components of the citric acid cycle failed to antagonize the inhibition by 5 pg./ml. of sulfathiazole in growth systems free from carbohydrates. Here, apparently, the citric acid cycle is probably operating at a reduced rate and the
4-AMIh’O-j-IMIDAZOLECARBOXAMIDE.
II
19
inhibition of growth must involve enzyme systems ot.her than those of the citric acid cycle and may be concerned with the amino acid metabolism . SUM~~ARY
AND CONCLUSION
Factors controlling the prevention of t.he accumulation of 4-amino-5imidazolecarboxamide during the growth of Escherichia wli R in 1% casein hydrolyzate medium containing 0.125% glucose were studied. p-hminobenzoic acid (PAnil) at cert,ain concentrations antagonized the inhibition of growth by sulfathiazole but failed to prevent the accumulation of the carboxamide in bot.h control and sulfathiazolecontaining systems. Citrovorum factor failed both to prevent t.he a+ cumulation of the amine and to antagonize the inhibition of growth. These facts may permit the conclusion t,hat PAPA antagonism to sulfathiazole inhibition of growt,h, and the metabolism of the carboxamide as a purine intermediary are independent processes, or the increase in amount of the accumulated carboxamide in t.hc presence of sulfathiazole may be due primarily to the effect of sulfathiazole on the citric acid cycle, and t.o inhibition of purine synthesis only secondarily to this primary inhibition. Aeration, both at the start. and during the incubation of the growth systems, prevents the accumulation of t.he carboxamide. This can be interpreted to indicate that the accumulation of the carboxamide is related to an impairment of the aerobic and the promotion of anaerobic metabolism. The increment in the amount of the carboxamide accumulated during sulfathiazole bacteriostasis may likewise indicate that sulfathiazole suppresses the aerobic and promotes the anaerobic metabolism of the system. Comparable to the effect obtained during aeration, the addition to the growth systems of mono- and dicarboxylic acids related t.o the citric arid cycle inhibits the accumulation of the carboxamide. These acids also antagonize t,he inhibition of growth by sulfathiazolc.
1. Fox,
C. L., JR., hoc. Sot. Ezpll. Riol. .Ued. 61, 102. (1942). M. It., ASD Fox, C. I,., *JR., J. Hiol. Chrm. 161.333 (1946). 3. SIIIVE, W., ACKER~~IASS, W. W., CORDOK, >I., GETAESDANER, M. E., ANI) E:AKIX, 11. E., J. Aw. Chenr. Sot. 69. 725 (1947). 4. MILLER, C. S., GURIS, S., ASI) WIISO~, n. W., Science 111, 654 (1950). 5. CONZELMAX, G. M.,JR.,MAsDEL. H. G., ASD SMITH, I'. K., Federation Proc. 11, 199 (1962). 2. STETITN,
20
SEVAG
AND
STEWART
6. SCHULI~AN, M. P., AND BUCHANAN, J. M., Federation Proc. J. Biol. Chem. 196, 513 (1952). 7. WILLIAMS, W. J., Federation Proc. 10, 270 (1951). 8. STEWART, R. C., AND SEVAG, M. G., Arch. Biochem. Biophys. 9. Fox, C. L., JR., J. Bact. 43, 68 (1942). 10. SEVAG, M. G., SHELBURNE, M., AND MIJDD, S., J. Bact. 49, 65 11. STEERS, E., AND SEVAG, M. G., Arch. Biochem. 24, 129 (1949). 12. SEVAG, M. G., GOTS, J. S., AND STEERS, E., in The Enzymes, p. 115. Academic Press, New York, 1950.
10, 244 (1951);
41, 9 (1952). (1945). Vol. I, Part
I,