Free Radical Biology & Medicine. Vol. 13, pp. 689-693, 1992 Printed in the USA. All rights reserved.
0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Brief Communication SYNTHETASE IS T H E S I T E O F PROTEIN A OF QUINOLINATE OXYGEN POISONING OF PYRIDINE NUCLEOTIDE COLI COENZYME SYNTHESIS IN ESCHERICHIA
BOZENA DRACZYNSKA-LUSIAK and OLEN R . BROWN Dalton Research Center and Departments of Biomedical Sciences and Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 6521 l, USA (Received 30 March 1992; Revised and Accepted 9 July 1992) A b s t r a c t - - D e novo biosynthesis of pyridine nucleotide coenzymes in Escherichia coli is initiated by an enzyme complex (quinolinate synthetase) containing protein B which converts L-aspartate into iminoaspartate and protein A, which then generates quinolinate on the pathway to the coenzymes. This complex has been shown to be poisoned by hyperbaric oxygen. 7'8 We performed assays made dependent on both proteins B and A versus only protein A, using cell-free extracts of hyperbaric-oxygen poisoned and aerobically grown cells. The specific activities were reduced by similar amounts of 68% and 60%, respectively, when measured in assays made dependent on enzymes B and A versus only protein A that was derived from oxygen-poisoned extract. Thus, protein A is the oxygen-sensitive component. Keywords--Oxygen toxicity, Oxidative stress, Enzyme inactivation, Pyridine nucleotide coenzymes, Escherichia coli, Free radicals
to air exposure. Brown and Seither 7 and Gardner and Fridovich 8 recently found that the complex containing protein A and B (quinolinate synthetase) is impaired in E. coli exposed to hyperbaric oxygen or aerobic paraquat, and Gardner and Fridovich s postulated on the basis of indirect evidence that protein A was the sensitive site. We now report by direct assay that protein A, and not protein B, is the site of inhibition by oxygen for the impaired biosynthesis of NAD and NADP in E. coil
INTRODUCTION
Exposure of Escherichia coli to 4.2 atm of partial pressure of oxygen results in a significant loss over time of the intracellular concentrations of the pyridine nucleotide coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP).I'z Exposure ofE. coli to aerobic paraquat also results in a loss of NAD and NADp.3 The decreased coenzymes have physiological significance. They become rate limiting for growth after about 1 h of oxidant stress in cultures supplemented with amino acids to prevent induction of stringency via oxidant-stress impairment of amino acid biosynthesis. 3 Evidence indicates that the pathway of synthesis of NAD and NADP (Fig. 1), from the intermediate quinolinate onward, is not significantly impaired by hyperbaric oxygen or aerobic paraquat. 4'5 Nasu et al. 6 have studied the pathway to quinolinate and reported that extracts of E. coli contain an enzyme complex composed of two proteins (A and B), required for synthesis of quinolinate, that is unstable
MATERIALS AND METHODS
Radiochemicals were purchased from the following sources: (U-~4C)D-fructose- 1,6-diphosphate, disodium salt (235 mCi/mmol) was from ICN Biomedicals, Inc. (Costa Mesa, CA) and L-(U-14C) aspartic acid (206 mCi/mmol) was from Amersham (Arlington Heights, IL). N,N-bis-(2-hydroxyethyl)glycine (bicine), quinolinic acid, dihydroxyacetone phosphate as the lithium salt, D-fructose-l,6-diphosphate, L-aspartic acid, aldolase from rabbit muscle, oxaloacetic acid and flavin adenine dinucleotide as the disodium salt were obtained from Sigma Chemical Co. (St. Louis, MO).
Address correspondence to: Dr. Olen R. Brown, Dalton Research Center, University of Missouri, Columbia, MO 65211. 689
690
B. DRACZYNSKA-LUSIAKand O. R. BROWN L-ASPARTATE ~
E-FAD -
H202
E-FADH~" ~
02
E. coli PROTEINB
°. i o H-C-H I
C=N-H
I.o
C,,0 -
[ IMINOASPARTATE ] ' DIHYDROXYACETONE E. coil PROTEINA
~C
PHOSPHATE
-~ 2H20+H3PO4
~ N - ~ C~'O \0
c~O \OQUINOLINATE
NAD+
:~ NADH
N!DP+~ - - - - - ~ NADPH Fig. 1. Biosynthesispathway for NAD(P) in Escherichia co/i.
Ion exchange resin AG l-X8 (200-400 mesh, hydroxide form) was purchased from Bio-Rad Laboratories (Richmond, CA), and Dowex 50W (X8, 50-100 mesh, hydrogen form) was obtained from Sigma. AG 1 resin was converted from the OH to the C1 form (in the column mode) by washing with two bed volumes of 1 M HC1. After converting, the resin was equilibrated to the desired starting conditions. The scintillation fluid was Bio-Safe II from Research Products International Corp. (Mount Prospect, IL). Escherichia coli K12 (CGSC strain #5073), obtained from the E. coli Genetic Stock Center, Yale University School of Medicine, New Haven, Connecticut, was grown in minimal salts-glucose medium. 6 Cultures were grown at 37°C with vigorous shaking in a Lab-Line Instruments, Inc. (Melrose Park, IL) orbit shaker at 300 rpm. Cultures in exponential growth at an absorbance of approximately 0.7 at 500 nm wavelength were pressurized in the pressure chamber of a Model 202 Amicon (W. R. Grace & Co., Beverly, MA) filtration device with stirring by magnetically coupled bar by adding oxygen to the 1 atm of air present. In different experiments, either 2.2 or 4.2 atm partial pressure of oxygen was obtained, and cultures were incubated for 30 min. Cell growth was stopped
by pouring cultures over crushed ice, and cells were collected by centrifugation at 7000 g for 10 min at 4°C. Cell pellets were washed with ice-chilled bicine (pH = 8.0), centrifugated at 10,000 g for 5 min, and resuspended in 1 mL of the aforementioned buffer. Cell-free extracts were prepared by sonic disruption of the cells using a Lab-Line Ultratip Labsonic System at 75 W power. Cells were sonicated for a total of 5 min, in 8-s intervals, and maintained below 8°C by intermittently submerging the test tube containing the cells in an ethanol-ice bath. 9 Extracts were clarifying at 16,000 g for 10 min, and the supernatants were stored at -70°C and used for quinolinate synthetase assays. Under these conditions, the quinolinate synthetase was stable prior to assay. Protein concentrations were determined with a Beckman Instruments, Inc. (Fullerton, CA) DU-64 spectrophotometer by the Bradford method l° using bovine serum albumin as the standard. Quinolinate synthetase assays were done in two different ways. One assay was dependent on both protein A and protein B activity. The other assay contained chemically generated iminoaspartate and thus was dependent only on protein A activity (Fig. 1). In each case the assay temperature was 25°C, and the total reaction volume was 500 uL with the same amount of cell-free extract protein (2.25 mg). Following the procedure proposed by Nasu et al., 6"11the reaction mixture dependent on only protein A contained bicine (pH = 8.0), 50 ~mol; (U-~4C) fructose diphosphate, 0.25 umol and 235 mCi/mmol; aldolase, 3.5 units to generate dihydroxyacetone-3-phosphate in situ; oxaloacetic acid, 2.5 ~mol, and (NH4)2SO4, 2.5 umol. Reactions were initiated by adding cell-free extracts to assay mixtures, were terminated after 5 min by adding 0.5 mL 20% perchloric acid followed by 1 umol of nonradioactive quinolinic acid as a cartier, and were chilled on ice for 15 min. The resulting protein precipitate was pelleted at 16,000 g for 5 min, and the supernatant was neutralized with 2 M KOH in an ice-water bath. After 30 min, the potassium perchlorate precipitate was removed by centrifugation at 16,000 g for 5 min at 4°C, and (~4C) quinolinate was isolated by the two-step ion-exchange chromatography system described by Chandler et a1.12 One unit of quinolinate synthetase activity is defined as the amount catalyzing the formation of 1 nmol of quinolinate per minute under the conditions of the assay (following the definition proposed by Gardner and FridovichS). It should be noted that three carbons from (UJ4C) D-fructose-l,6-disphosphate (FDP) are incorporated into quinolinate. The second enzyme assay procedure was made dependent on both protein A and protein B activities of
02 poisoning of NAD(P) synthesis
I ~
1.0
o~
0.9
o
i
i
i O~ O
O.8 E 0.7 r" 0.6
0
I
i
zx
°
o~
0 0
691
o Control pO 2 =0.2 atm 0.5
0 Z < rng~Om r'
0.3
o
F-
90
l--
70
S Z
50
to hyperbaric oxygen since 2.2 atm of oxygen pressure minimally inhibited E. coli growth but the enzyme activity was decreased about 40%. Gardner and Fridovich8 have reported that the enzyme activity catalyzed by the quinolinate synthetase complex is diminished by exposure of E. coli to 4.2 arm of oxygen. They suggested that protein A was the sensitive site and provided preliminary evidence that it is an (Fe-S)4-containing enzyme. This evidence included the presence of the -cys-w-x-cys-y-z-cys-sequence (characteristic of (Fe-S)4-containing proteins) and the fact that a, a~-dipyridyl and 1,10-phenanthroline blocked the reactivation observed during anaerobic incubation of oxygen-inactivated enzyme, s
,
I-I,~+
6.8
%
•
UJ
%
4O 3O
. I
I
O.2 OXYGEN
2.2 PRESSURE (atm)
o I
4.2
Fig. 3. The decline (in %) of quinolinate activity in assays with chemically generated iminoaspartate, caused by hyperbaric oxygen (points are average of triplicate determinations).
02 poisoning of NAD(P) synthesis
They also concluded that the oxidant inactivation appeared to be caused by 02 and was not directly mediated by superoxide or H202. The protein A component of the quinolinate synthetase of E. coli thus appears to share three characteristics with another oxidant-sensitive enzyme, dihydroxyacid dehydratase, ~3-16 from this organism: high sensitivity to inactivation by oxidant stress, function as a dehydratase, and the presence of an iron-sulfur cluster. Acknowledgement--This research was supported in part by DHHS NIEHS R01-02566. REFERENCES
1. Brunker, R. L.; Brown, O. R. Effects ofhyperoxia on oxidized and reduced NAD and NADP concentrations in Escherichia coil Microbios 4:193-203; 1971. 2. Brown, O. R.; Song, C. S. Pyridine nucleotide coenzyme biosynthesis: A cellular site of oxygen toxicity. Biochem. Biophys. Res. Comm. 93:172-178; 1980. 3. Heitkamp, M.; Brown, O. R. Inhibition of NAD biosynthesis by paraquat in Escherichia coll. Biochim. Biophys. Acta 676:345-349; 1981. 4. Brown, O. R.; Seither, R. L. Paraquat toxicity and pyridine nucleotide coenzyme synthesis: A data correction. Free Rad. Biol. Med. 8:113-116; 1990. 5. Gardner, P. R.; Fridovich, I. Quinolinate phosporibosyl transferase is not the oxygen-sensitive site of nicotinamide adenine dinucleotide biosynthesis. Free R a d Biol. Med. 8:117-119; 1990.
693
6. Nasu, S.; Wicks, F. D.; Gholson, R. K. L-aspartate oxidase, a newly discovered enzyme of Escherichia coli, is the B-protein of quinolinate synthetase. J. Biol. Chem. 257:626-632; 1982. 7. Brown, O. R.; Seither, R. L. Paraquat inhibits NAD biosynthesis at the quinolinic acid synthetase site. Med. Sci. Res. 17:819820; 1989. 8. Gardner, P. R.; Fridovich, 1. Quinolinate synthetase: The oxygen-sensitive site of de novo NAD(P) biosynthesis. Arch. Biochem. Biophys. 284:106-111; 1991. 9. Chandler, J. L. R.; Gholson, R. K. Studies on the biosynthesis of NAD in Escherichia coli. III. Precursors ofquinolinic acid in vitro. Biochim. Biophys. Acta 264:311-318; 1972. 10. Bradford, M. A. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254; 1976. 11. Nasu, S.; Gholson, R. K. Replacement of the B protein requirement of the E. coli quinolinate synthetase system by chemically-generated immunoaspartate. Biochem. Biophys. Res. Comm. 101:533-539; 1981. 12. Chandler, J. L. R.; Gholson, R. K. Studies on the de novo biosynthesis of NAD in Escherichia coli: II. Quantitative method for isolating quinolinic acid from biological materials. Anal. Biochem. 48:529-535; 1972. 13. Seither, R. L.; Brown, O. R. Induction of stringency by hyperoxia in Escherichia coli. Cell. Mol. Biol. 28:285-291; 1982. 14. Brown, O. R.; Yein, F. Dihydroxyacid dehydratase: The site of hyperbaric oxygen poisoning in branch-chain amino acid biosynthesis. Biochem. Biophys. Res. Comm. 85:219-224; 1978. 15. Kuo, C. F.; Mashino, T.; Fridovich, I. a,fl-dihydroxyisovolerate dehydratase a superoxide-sensitive enzyme. J. Biol. Chem. 262:4724-4727; 1987. 16. Flint, D. H.; Emptage, M. H. Dihydroxyacid dehydratase-isolation, characterization as Fe-S proteins, and sensitivity to inactivation by 02. In: Barak, Z.; Chapman, D. M.; Schloss, J. V., eds. Biosynthesis o f branched chain amino acids New York: V.C.H. Weinheim; 1990:285-314.
0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Brief Communication SYNTHETASE IS T H E S I T E O F PROTEIN A OF QUINOLINATE OXYGEN POISONING OF PYRIDINE NUCLEOTIDE COLI COENZYME SYNTHESIS IN ESCHERICHIA
BOZENA DRACZYNSKA-LUSIAK and OLEN R . BROWN Dalton Research Center and Departments of Biomedical Sciences and Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 6521 l, USA (Received 30 March 1992; Revised and Accepted 9 July 1992) A b s t r a c t - - D e novo biosynthesis of pyridine nucleotide coenzymes in Escherichia coli is initiated by an enzyme complex (quinolinate synthetase) containing protein B which converts L-aspartate into iminoaspartate and protein A, which then generates quinolinate on the pathway to the coenzymes. This complex has been shown to be poisoned by hyperbaric oxygen. 7'8 We performed assays made dependent on both proteins B and A versus only protein A, using cell-free extracts of hyperbaric-oxygen poisoned and aerobically grown cells. The specific activities were reduced by similar amounts of 68% and 60%, respectively, when measured in assays made dependent on enzymes B and A versus only protein A that was derived from oxygen-poisoned extract. Thus, protein A is the oxygen-sensitive component. Keywords--Oxygen toxicity, Oxidative stress, Enzyme inactivation, Pyridine nucleotide coenzymes, Escherichia coli, Free radicals
to air exposure. Brown and Seither 7 and Gardner and Fridovich 8 recently found that the complex containing protein A and B (quinolinate synthetase) is impaired in E. coli exposed to hyperbaric oxygen or aerobic paraquat, and Gardner and Fridovich s postulated on the basis of indirect evidence that protein A was the sensitive site. We now report by direct assay that protein A, and not protein B, is the site of inhibition by oxygen for the impaired biosynthesis of NAD and NADP in E. coil
INTRODUCTION
Exposure of Escherichia coli to 4.2 atm of partial pressure of oxygen results in a significant loss over time of the intracellular concentrations of the pyridine nucleotide coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP).I'z Exposure ofE. coli to aerobic paraquat also results in a loss of NAD and NADp.3 The decreased coenzymes have physiological significance. They become rate limiting for growth after about 1 h of oxidant stress in cultures supplemented with amino acids to prevent induction of stringency via oxidant-stress impairment of amino acid biosynthesis. 3 Evidence indicates that the pathway of synthesis of NAD and NADP (Fig. 1), from the intermediate quinolinate onward, is not significantly impaired by hyperbaric oxygen or aerobic paraquat. 4'5 Nasu et al. 6 have studied the pathway to quinolinate and reported that extracts of E. coli contain an enzyme complex composed of two proteins (A and B), required for synthesis of quinolinate, that is unstable
MATERIALS AND METHODS
Radiochemicals were purchased from the following sources: (U-~4C)D-fructose- 1,6-diphosphate, disodium salt (235 mCi/mmol) was from ICN Biomedicals, Inc. (Costa Mesa, CA) and L-(U-14C) aspartic acid (206 mCi/mmol) was from Amersham (Arlington Heights, IL). N,N-bis-(2-hydroxyethyl)glycine (bicine), quinolinic acid, dihydroxyacetone phosphate as the lithium salt, D-fructose-l,6-diphosphate, L-aspartic acid, aldolase from rabbit muscle, oxaloacetic acid and flavin adenine dinucleotide as the disodium salt were obtained from Sigma Chemical Co. (St. Louis, MO).
Address correspondence to: Dr. Olen R. Brown, Dalton Research Center, University of Missouri, Columbia, MO 65211. 689
690
B. DRACZYNSKA-LUSIAKand O. R. BROWN L-ASPARTATE ~
E-FAD -
H202
E-FADH~" ~
02
E. coli PROTEINB
°. i o H-C-H I
C=N-H
I.o
C,,0 -
[ IMINOASPARTATE ] ' DIHYDROXYACETONE E. coil PROTEINA
~C
PHOSPHATE
-~ 2H20+H3PO4
~ N - ~ C~'O \0
c~O \OQUINOLINATE
NAD+
:~ NADH
N!DP+~ - - - - - ~ NADPH Fig. 1. Biosynthesispathway for NAD(P) in Escherichia co/i.
Ion exchange resin AG l-X8 (200-400 mesh, hydroxide form) was purchased from Bio-Rad Laboratories (Richmond, CA), and Dowex 50W (X8, 50-100 mesh, hydrogen form) was obtained from Sigma. AG 1 resin was converted from the OH to the C1 form (in the column mode) by washing with two bed volumes of 1 M HC1. After converting, the resin was equilibrated to the desired starting conditions. The scintillation fluid was Bio-Safe II from Research Products International Corp. (Mount Prospect, IL). Escherichia coli K12 (CGSC strain #5073), obtained from the E. coli Genetic Stock Center, Yale University School of Medicine, New Haven, Connecticut, was grown in minimal salts-glucose medium. 6 Cultures were grown at 37°C with vigorous shaking in a Lab-Line Instruments, Inc. (Melrose Park, IL) orbit shaker at 300 rpm. Cultures in exponential growth at an absorbance of approximately 0.7 at 500 nm wavelength were pressurized in the pressure chamber of a Model 202 Amicon (W. R. Grace & Co., Beverly, MA) filtration device with stirring by magnetically coupled bar by adding oxygen to the 1 atm of air present. In different experiments, either 2.2 or 4.2 atm partial pressure of oxygen was obtained, and cultures were incubated for 30 min. Cell growth was stopped
by pouring cultures over crushed ice, and cells were collected by centrifugation at 7000 g for 10 min at 4°C. Cell pellets were washed with ice-chilled bicine (pH = 8.0), centrifugated at 10,000 g for 5 min, and resuspended in 1 mL of the aforementioned buffer. Cell-free extracts were prepared by sonic disruption of the cells using a Lab-Line Ultratip Labsonic System at 75 W power. Cells were sonicated for a total of 5 min, in 8-s intervals, and maintained below 8°C by intermittently submerging the test tube containing the cells in an ethanol-ice bath. 9 Extracts were clarifying at 16,000 g for 10 min, and the supernatants were stored at -70°C and used for quinolinate synthetase assays. Under these conditions, the quinolinate synthetase was stable prior to assay. Protein concentrations were determined with a Beckman Instruments, Inc. (Fullerton, CA) DU-64 spectrophotometer by the Bradford method l° using bovine serum albumin as the standard. Quinolinate synthetase assays were done in two different ways. One assay was dependent on both protein A and protein B activity. The other assay contained chemically generated iminoaspartate and thus was dependent only on protein A activity (Fig. 1). In each case the assay temperature was 25°C, and the total reaction volume was 500 uL with the same amount of cell-free extract protein (2.25 mg). Following the procedure proposed by Nasu et al., 6"11the reaction mixture dependent on only protein A contained bicine (pH = 8.0), 50 ~mol; (U-~4C) fructose diphosphate, 0.25 umol and 235 mCi/mmol; aldolase, 3.5 units to generate dihydroxyacetone-3-phosphate in situ; oxaloacetic acid, 2.5 ~mol, and (NH4)2SO4, 2.5 umol. Reactions were initiated by adding cell-free extracts to assay mixtures, were terminated after 5 min by adding 0.5 mL 20% perchloric acid followed by 1 umol of nonradioactive quinolinic acid as a cartier, and were chilled on ice for 15 min. The resulting protein precipitate was pelleted at 16,000 g for 5 min, and the supernatant was neutralized with 2 M KOH in an ice-water bath. After 30 min, the potassium perchlorate precipitate was removed by centrifugation at 16,000 g for 5 min at 4°C, and (~4C) quinolinate was isolated by the two-step ion-exchange chromatography system described by Chandler et a1.12 One unit of quinolinate synthetase activity is defined as the amount catalyzing the formation of 1 nmol of quinolinate per minute under the conditions of the assay (following the definition proposed by Gardner and FridovichS). It should be noted that three carbons from (UJ4C) D-fructose-l,6-disphosphate (FDP) are incorporated into quinolinate. The second enzyme assay procedure was made dependent on both protein A and protein B activities of
02 poisoning of NAD(P) synthesis
I ~
1.0
o~
0.9
o
i
i
i O~ O
O.8 E 0.7 r" 0.6
0
I
i
zx
°
o~
0 0
691
o Control pO 2 =0.2 atm 0.5
0 Z < rng~Om r'
0.3
o
F-
90
l--
70
S Z
50
to hyperbaric oxygen since 2.2 atm of oxygen pressure minimally inhibited E. coli growth but the enzyme activity was decreased about 40%. Gardner and Fridovich8 have reported that the enzyme activity catalyzed by the quinolinate synthetase complex is diminished by exposure of E. coli to 4.2 arm of oxygen. They suggested that protein A was the sensitive site and provided preliminary evidence that it is an (Fe-S)4-containing enzyme. This evidence included the presence of the -cys-w-x-cys-y-z-cys-sequence (characteristic of (Fe-S)4-containing proteins) and the fact that a, a~-dipyridyl and 1,10-phenanthroline blocked the reactivation observed during anaerobic incubation of oxygen-inactivated enzyme, s
,
I-I,~+
6.8
%
•
UJ
%
4O 3O
. I
I
O.2 OXYGEN
2.2 PRESSURE (atm)
o I
4.2
Fig. 3. The decline (in %) of quinolinate activity in assays with chemically generated iminoaspartate, caused by hyperbaric oxygen (points are average of triplicate determinations).
02 poisoning of NAD(P) synthesis
They also concluded that the oxidant inactivation appeared to be caused by 02 and was not directly mediated by superoxide or H202. The protein A component of the quinolinate synthetase of E. coli thus appears to share three characteristics with another oxidant-sensitive enzyme, dihydroxyacid dehydratase, ~3-16 from this organism: high sensitivity to inactivation by oxidant stress, function as a dehydratase, and the presence of an iron-sulfur cluster. Acknowledgement--This research was supported in part by DHHS NIEHS R01-02566. REFERENCES
1. Brunker, R. L.; Brown, O. R. Effects ofhyperoxia on oxidized and reduced NAD and NADP concentrations in Escherichia coil Microbios 4:193-203; 1971. 2. Brown, O. R.; Song, C. S. Pyridine nucleotide coenzyme biosynthesis: A cellular site of oxygen toxicity. Biochem. Biophys. Res. Comm. 93:172-178; 1980. 3. Heitkamp, M.; Brown, O. R. Inhibition of NAD biosynthesis by paraquat in Escherichia coll. Biochim. Biophys. Acta 676:345-349; 1981. 4. Brown, O. R.; Seither, R. L. Paraquat toxicity and pyridine nucleotide coenzyme synthesis: A data correction. Free Rad. Biol. Med. 8:113-116; 1990. 5. Gardner, P. R.; Fridovich, I. Quinolinate phosporibosyl transferase is not the oxygen-sensitive site of nicotinamide adenine dinucleotide biosynthesis. Free R a d Biol. Med. 8:117-119; 1990.
693
6. Nasu, S.; Wicks, F. D.; Gholson, R. K. L-aspartate oxidase, a newly discovered enzyme of Escherichia coli, is the B-protein of quinolinate synthetase. J. Biol. Chem. 257:626-632; 1982. 7. Brown, O. R.; Seither, R. L. Paraquat inhibits NAD biosynthesis at the quinolinic acid synthetase site. Med. Sci. Res. 17:819820; 1989. 8. Gardner, P. R.; Fridovich, 1. Quinolinate synthetase: The oxygen-sensitive site of de novo NAD(P) biosynthesis. Arch. Biochem. Biophys. 284:106-111; 1991. 9. Chandler, J. L. R.; Gholson, R. K. Studies on the biosynthesis of NAD in Escherichia coli. III. Precursors ofquinolinic acid in vitro. Biochim. Biophys. Acta 264:311-318; 1972. 10. Bradford, M. A. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254; 1976. 11. Nasu, S.; Gholson, R. K. Replacement of the B protein requirement of the E. coli quinolinate synthetase system by chemically-generated immunoaspartate. Biochem. Biophys. Res. Comm. 101:533-539; 1981. 12. Chandler, J. L. R.; Gholson, R. K. Studies on the de novo biosynthesis of NAD in Escherichia coli: II. Quantitative method for isolating quinolinic acid from biological materials. Anal. Biochem. 48:529-535; 1972. 13. Seither, R. L.; Brown, O. R. Induction of stringency by hyperoxia in Escherichia coli. Cell. Mol. Biol. 28:285-291; 1982. 14. Brown, O. R.; Yein, F. Dihydroxyacid dehydratase: The site of hyperbaric oxygen poisoning in branch-chain amino acid biosynthesis. Biochem. Biophys. Res. Comm. 85:219-224; 1978. 15. Kuo, C. F.; Mashino, T.; Fridovich, I. a,fl-dihydroxyisovolerate dehydratase a superoxide-sensitive enzyme. J. Biol. Chem. 262:4724-4727; 1987. 16. Flint, D. H.; Emptage, M. H. Dihydroxyacid dehydratase-isolation, characterization as Fe-S proteins, and sensitivity to inactivation by 02. In: Barak, Z.; Chapman, D. M.; Schloss, J. V., eds. Biosynthesis o f branched chain amino acids New York: V.C.H. Weinheim; 1990:285-314.
