The role of glutathione in escherichia coli

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E ROLE OF

GLUTATHIONE

IN ESCHERICHIA COLi

P. APONTOWEIL

P1798

4226

C10053

85545

THE ROLE OF

GLUTATHIONE IN ESCHERICHIA COLI

PROEFSCHRIFT

ter verkrijging van de graad van doctor in de techni-sche wetenschappen aan de Technitechni-sche Hogeschool Delft, op gezag van de rector magnificus prof. dr. ir. H. van Bekkum, voor een commissie aangewezen door het college van dekanen te verdedigen op

woensdag 2 juni 1976 te 16.00 uur

door

PETER APONTOWEIL scheikundig ingenieur

geboren te Batavia

1976 ^19^ ^^^^

DIT PROEFSCHRIFT IS GOEDGEKEURD DOOR DE PROMOTOR PROF. W. BERENDS

CONTENTS

GENERAL INTRODUCTION V History and Occurrence v

Metabolism V Biological functions viil

Aim of the investigation and method of approach xi

References xii PAPERS

I. Apontoweil, P. and Berends, W. (1975),

Glutathione biosynthesis in Escherichia coli K 12 — Properties of the enzymes and regulation.

Biochim. Biophys. Acta 399, 1-9 II. Apontoweil, P. and Berends, W. (1975),

Isolation and initial characterization of glutathione-defi-cient mutants of Escherichia coli K 12.

Biochim. Biophys. Acta 399, 10-22 in. Apontoweil, P. and Berends, W. (1975),

Mapping of gshA, a gene for the biosynthesisof glutathione in Escherichia coli K 12.

Molec. Gen. Genet. 141, 91-95 SUMMARY AND CONCLUSIONS

SAMENVATTING EN CONCLUSIES NAWOORD

V GENERAL INTRODUCTION

History and Occurrence

Glutathione is the tripeptide 7-L-glutamyl-L-cysteinylglycine(GSH). It was first described by De Rey-Pailhade in 1888 as a crude substance obtained from yeast, and capable of reducing sulphur to H2S (1). In 1921 Hopkins isolated glutathione in a crystalline form from yeast and originally regarded the formula as a dipeptide of the two amino acids, glutamic acid and cysteine (2). Subsequent studies by Hopkins (3) and by Kendall et al. (4) led to the conclusion that the molecule was a tripeptide containing glutamic acid, cysteine and glycine. The correct structure (I) was confirmed by the titration studies of Pirie and Pinhey (5) and by Harington and Mead (6), who compared the properties of the synthetic and the natural tripeptide.

NH2 CHj SH

I I

HOOCCHCH2 CH2 CONHCHCONHCH2 C O O H

Glutathione appears to occur in all kinds of cells. It has been detected in animal tissues, plants and microorganisms as the major low-molecular-weight thiol. Glutathione occurs in concentrations of 1-100 x

10"* M (7-9). It is present in yeast (7), liver (10) and eye lens (11, 12) in relative abundance. The amounts present in a given tissue are subject to variation with the growth and nutritional state of, or any stress applied to the organism (e.g. hormonal treatment, diets, drug administration, irradiation). Most of the hundreds of entries under Glutathione each year in Chemical Abstracts relate to the effects of such influences on the GSH-level or the consequences of adding GSH to some enzyme system.

Little information is available about the occurrence of glutathione in bacteria. Its presence has been reported in Staphylococcus aureus (13) and in Escherichia coli (14, 15). Glutathione represents at least 95% of the acid-soluble sulphur compounds in E. coli, accounting for approx. 25% of the total sulphur content of the cell (16).

Metabolism

The biosynthesis of glutathione from glutamic acid, cysteine and glycine was first observed in vitro with rat liver slices by Braunstein et al.

VI

in 1948 (17). Bloch and co-workers demonstrated the synthesis of glutathione in cell-free systems of liver and established that glutathione synthesis occurs in two enzymatic steps (18, 19, 20). The first step (II) consists in the formation of a peptide bond between glutamic acid and cysteine, catalyzed by 7-glutamylcysteine synthetase. In the second step (III) the product 7-glutamylcysteine forms a peptide bond with glycine, catalyzed by glutathione synthetase. ATP and Mg^"^ are needed in both steps.

glutamic acid + cysteine + ATP *• 7-glutamylcysteine + ADP + Pi (II) 7-glutamylcysteine + glycine + ATP ^ glutathione + ADP + Pi (III) The same scheme of synthesis appears to be followed in kidney (21), erythrocytes (22, 23), various plants (24) and yeast (25, 26). The bio-synthesis of glutathione in extracts of E. coli has been studied by Samuels (27). This report only describes the factors required for the overall synthesis from the three component amino acids. Investigations on purified preparations of 7-glutamylcysteine synthetase obtained from rat kidney (28), rat liver and toad Hver (29) indicate that an enzyme-bound 7-glutamyl phosphate is formed as an intermediate stage. The formation of an enzyme-bound acyl phosphate has also been shown in the reaction catalyzed by glutathione synthetase. The dipeptidyl phosphate intermediate has been isolated from a reaction mixture con-taining enzyme from yeast, dipeptide, Mg^* and ATP (26).

Most of the glutathione in living cells is in the reduced form (GSH). The oxidized form, glutathione disulphide (GSSG), has been detected in very small amounts (0-2% of total glutathione) (30, 31). It is uncertain whether at least part of these amounts are artefacts due to auto-oxida-tion of GSH during the assay procedure. The oxidaauto-oxida-tion of GSH to GSSG may proceed nonenzymically:

2 GSH ^ GSSG + 2H^ + 2 e

A few systems are known in which the oxixation of GSH is catalyzed by an enzyme. Dehydroascorbic acid reductase, an enzyme present in plants (32-34), catalyses the reaction:

VII

Glutathione peroxidase catalyses the oxidation of GSH by hydrogen peroxide. The enzyme occurs in several animal tissues (35), but attention has been focused on its role in protecting hemoglobin in erythrocytes (36, 37). Glutathione disulphide may also be formed by thiol-disulphide interchange reactions. In these reactions disulphides (symmetrical, RSSR or mixed with glutathione, RSSG) can be reduced by GSH:

RSSR + GSH ^ GSSR + RSH

GSSR + GSH ^ GSSG + RSH

The occurrence of the mixed disulphides of glutathione with coenzyme A (38-40) and of glutathione with cysteine (41, 42) has been demon-strated in biological materials. The reactions of GSH with disulphides can take place spontaneously but are also catalyzed by enzymes (39, 43-46), which have been named transhydrogenases (43) or rather thioltrans-ferases (47).

High concentrations of GSSG will be prevented by the action of the enzyme glutathione reductase, which is widely distributed (48-51) and catalyses the reaction:

GSSG -I- NADPH + H+—>2 GSH + NADP+

The preferential hydrogen donor is NADPH but NADH can also be used (52, 53). NADPH is generated from NADP* by oxidation of glucose-6-phosphate and of 6-phospho-gluconate. Hence the reduction of GSSG is thought to be coupled to the hexose monophosphate shunt pathway (54-56).

The pathway for the degradation of glutathione involves the removal of its 7-glutamyl group as the first step. The resulting dipeptide cysteinylglycine is then hydrolysed to glycine and cysteine (57, 58). The 7-glutamyl group removed from GSH may be converted in three ways; to glutamate by hydrolyses (59), to 5-oxoproline by cycHzation (60, 61) and to 7-glutamylpeptide by transfer to another amino acid or peptide (62-65). 7-Glutamyl transfer is catalyzed by the enzyme 7-glutamyl transpeptidase. The presence of this enzyme has been demonstrated in various animal tissues (60, 65), erythrocytes (22), plants (66) and bacteria (67). The highest activities of 7-glutamyl transpeptidase are found in the kidney. It has been purified from the kidney and studied intensively by Meister and co-workers (65, 68). It should be noted that

VIII

E. coli can grow with glutathione as the sole sulphur source (16, 69). Roberts et al. (16) observed that endogenous glutathione can serve as emergency supply of sulphur when no sulphur is available from the growth medium.

Glutathione synthesis is controlled by feedback inhibition. It has been found in erythrocytes (22), rat kidney (70) and rat liver (29) that 7-glutamyl-cysteine synthetase is inhibited by GSH. The enzyme from erythrocytes also appears to be inhibited by NADH; the rat-liver enzyme is Ukewise inhibited by GSSG, NADPH and NADP*, and is activated by glycine. The second synthesizing enzyme from erythrocytes, glutathione synthetase, is inhibited by ADP (22). Kinetic studies on both synthe-sizing enzymes from erythrocytes have revealed that the rate of 7-gIuta-mylcysteine formation exceeds that of GSH formation by a factor of 2 - 4 (71-73). As against this, the occurrence of 7-glutamylcysteine has never been reported. More complete kinetic investigations, such as those now being carried out by Wendell and co-workers (74, 75) and by Meister and co-workers (70), may provide further insight into the mech-anism of regulation of glutathione biosynthesis.

Biological Functions

The biological functions of glutathione are not fully understood. In studying the function of glutathione in cell metabolism the sulfhydryl group is considered to be very important. The sulfhydryl group in glutathione is reactive, undergoing oxidation and alkylation, forming mercaptides with metal ions and reacting with double bonds by addition. The reactivity of a small thiol molecule did indicate that glutathione may have a protective function. It may protect various molecules, including proteins, by preventing their oxidation or by protecting them against toxic heavy metals. This theory about the physiological role of glutathione was first described by Barron in 1951 (76). There is no doubt that GSH has such effects in isolated systems (77-79). But it is difficult to show whether such protection is necessary in the intact cell. The importance of the protective action of intracellular glutathione has so far been well established only in erythrocytes. GSH is present in erythrocytes in amounts of 2 - 2,5 mM (80) and accounts for almost all the non-protein thiol. It has been found to be the main agent for protecting hemoglobin against hydrogen peroxide (36). Together with glutathione peroxidase it forms a system which can decompose hydrogen peroxide according to the following reaction:

IX

2GSH + H2O2 >GSSG + 2H2 0

GSH can be regenerated from GSSG by glutathione reductase. Catalase is also present in erythrocytes, but plays a less significant role at the low H2O2-concentrations (37, 81). Deficiences in glutathione biosynthesis have been observed in erythrocytes (71, 82, 83). These abnormalities concern a lack in one of the two synthesizing enzymes and result in a very low erythrocyte GSH-level (5-10% of normal). Patients whose erythrocytes show this deficiency suffer from a mild hemolytic anemia, but a hemolytic crisis may be induced by administering oxidative drugs (82). These findings indicate that GSH is beneficial to the red cell, probably by preserving the sulfhydryl groups of proteins (84, 85).

Glutathione plays a different kind of protective role in the detoxi-cation of foreign compounds by forming mercapturic acids (86-88). Mercapturic acids are S-substituted N-acetylcysteines:

R - S - CH2 CH(NHC0CH3 )COOH

These substances are excreted as detoxication products from foreign compounds (such as aryl and alkyl halides, nitroalkanes, aromatic hydro-carbons, halogenonitrobenzenes, sulphonamides, aj3-unsaturated com-pounds) administered to animals (88). The following pathway is sug-gested (88, 89):

RX + GSH > R-SG > R-S-Cys > R-S-Cys(N-acetyl) The first step in mercapturic acid biosynthesis is the conjugation of GSH with the foreign compound. Enzymes have been described which catalyse in vitro conjugation reactions. The enzymes are called gluta-thione S-transferases and are found mainly in liver extracts (90-94). Nonenzymic reactions also contribute to conjugations (88). The gluta-thione conjugate is thought to be metabolized further by cleavage of the glutamate and glycine residues. The last step involves an acylation of the free amino group of the cysteinyl residue by coenzyme A. The amounts of the mercapturic acids excreted, often represent only a small propor-tion (1-30%) of the dose administered (89, 94). Mercapturic acid forma-tion thus seems to represent only a minor route for the eliminaforma-tion of foreign compounds. No reports are known about the route in micro-organisms.

It has been suggested that the breakdown of glutathione is part of a mechanism for the transport of amino acids across cell membranes (63, 95). The function of glutathione is to supply the carrier, the 7-glutamyl

X

group. The enzyme 7-glutamyl transpeptidase catalyses the transfer of the 7-glutamyl group to an amino acid on the membrane (62-65). The 7-glutamyl-amino acid transported inside the cell is then converted into free amino acid and 5-oxoproline by 7-glutamyl cyclotransferase (61, 96, 97). Cysteinylglycine is cleaved by a peptidase to free cysteine and glycine (57, 58). A newly discovered enzyme (5-oxoprolinase) catalyses the conversion of 5-oxoproline to glutamate, requiring ATP (98). The free glutamate, cysteine and glycine will be utilized again for the biosyn-thesis of glutathione. The six reactions form the 7-glutamyl cycle and its functioning is now being strongly supported by Meister (98). The pres-ence of the six enzymes has been demonstrated in the kidney and brain (99). 7-Glutamyl transpeptidase is a membrane-bound enzyme and is present in the kidney in abundance. In other tissues 7-glutamyl trans-peptidase activity is very low (64, 100). The enzyme is infrequently en-countered in bacteria (67). The amino acid transport mediated by the 7-glutamyl cycle has a relatively high energy requirement. A mechanism of this type may be specifically applicable to some tissues but it could not be generahzed.

There are several enzyme-catalyzed reactions in which glutathione is involved as a coenzyme. The enzymes for which it has been found im-possible to replace glutathione by other thiols include formaldehyde dehydrogenase and the glyoxalase system. Formaldehyde dehydrogenase catalyses the oxidation of formaldehyde to formic acid:

H2 CO + NAD^ + H2 O > HCOOH + NADH + H+

The enzyme is present in liver (101), lens (102) and microorganisms (103, 104). The substrate is believed to be the hemimercaptal (GS-CH2OH) formed nonenzymically between formaldehyde and glutathione. The similar oxidation of acetaldehyde utilises a different enzyme and is found to be independent of GSH. The elimination of formaldehyde is also known to occur via tetrahydrofolate. The glyoxalase enzyme system consists of two enzymes, glyoxalase I and glyoxalase II, which respectively catalyse the following reactions (105,

106):

CH3COCHO + GSH > CH3CHOHCO - SG

CH3CHOHCO - SG

>

CH3CHOHCOOH + GSH

XI

Glyoxalase I mediates the isomerization of the hemimercaptal, formed initially by a nonenzymic reaction between glutathione and methylglyo-xal (107). The isomerization product S-lactoylglutathione is then hydro-lyzed to D-lactate. Methylglyoxal appears to be a toxic compound for it inhibits in particular protein synthesis (108-110). A few reactions are known which produce methylglyoxal. The enzymic conversion of dihydroxyacetone phosphate (a glycolytic intermediate) to methyl-glyoxal has been shown in bacteria (111, 112). The pathway leading via D-lactate to pyruvate represents a bypass of the normal glucose break-down. The degradation of threonine to lactic acid can occur in the higher animals and also in bacteria. Decarboxylation of threonine results in the formation of aminoacetone, which can be deaminated to give methyl-glyoxal (113-115). Both pathways are considered to be curious and their physiological significance is uncertain. The glyoxalase reaction has often been used for the determination of glutathione, since glutathione is a highly specific cofactor. Under good assay conditions the rate of conver-sion of methylglyoxal to lactic acid is proportional to the glutathione concentration (116). Glutathione may also be determined with glyoxalase I alone. S-lactoylglutathione formation can be followed from the increase in absorbance at 240 mn (117).

It is well-known that sulfhydryl compounds act in biological sys-tems as radioprotectors (118, 119). Representing the major component of the low-molecular-weight thiols in the cell, glutathione may exert a protective effect against ionizing radiation. A mechanism by which SH compounds can reduce cellular damage is that of scavenging radicals. The radicals include both the damaging (mainly free water radicals) or the damaged (radicals of the organic cell constituents). Hydrogen atom transfer of the SH groups to the radiation-induced radicals results in less reactive sulphur radicals. In several studies the radiosensitivity of bacterial and mammalian cells has been found to be dependent on their SH con-tent. In general, the higher the SH content, the greater the radioresistan-cy (120-122). Incubation of bacterial cells with p-hydroxymercuriben-zoate to bind cellular SH groups results in sensitization to X-rays (120,

123).

Aim of the investigation and method of approach

Much effort has been expended on elucidating the role of gluta-thione. The universahty of its presence suggests that glutathione may play a fundamental role common to all cells. One of the proposed

XII

biological functions, which could justify its presence in all kinds of cells, may be the protection of sulfhydryl groups No convincing evidence has been provided that glutathione in vivo is essential for protection As yet, the only indication that the protective action of glutathione is important in the intact cell has come from studies on erythrocytes with a distur-bance in GSH biosynthesis However, these erythrocytes were not entirely devoid of GSH and the question remains as to what other func-tion could require the small quantity of GSH (at least 5% of normal) It IS still beheved that a complete absence of GSH would be lethal

One difficulty in elucidating the role of glutathione is the absence of a genetic model which has a complete lack of glutathione and whose other cell functions are unaffected In my opinion, the combination of the results of both a genetic study and a study on the regulation of gluta-thione biosynthesis, would reveal what endogenous glutagluta-thione means for a cell under different conditons

The aim of the present investigation was to isolate and characterize mutants of E coli with a complete lack of GSH, and to investigate the factors which determine the intracellular concentration of glutathione. The results of both hnes of investigation are given in this thesis

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Reprinted from 1

Biochimica et Biophysica Acta, 399 (1975) 1—9

© Elsevier Scientific Publishing Company, Amsterdam — Printed in The Netherlands

BBA 27689

GLUTATHIONE BIOSYNTHESIS IN ESCHERICHIA COLI K 12 PROPERTIES OF THE ENZYMES AND REGULATION

P. APONTOWEIL and W. BERENDS

Biochemical and Biophysical Laboratory, Delft University of Technology, Julianalaan 67, Delft (The Netherlands)

(Received February 21st, 1975)

Summary

The synthesis of glutathione in Escherichia coli K 12 was studied in crude, cell-free extracts. The pH optima and the apparent K^ values for the substrates have been determined for both synthesizing enzymes, 7-glutamylcysteine syn-thetase and glutathione synsyn-thetase.

7-Glutamylcysteine synthetase was found to be approximately twice as active as glutathione synthetase. In a growing culture, the cellular level of GSH showed a considerable increase up to 6.6 lumol per ml cell pellet in the station-ary growth phase. GSSG was not detectable. The levels of the enzymes re-mained constant, indicating that glutathione biosynthesis depends at least in the beginning on the availability of the component amino acids. The pathway is controlled by feedback inhibition and not by repression.

Introduction

The tripeptide glutathione (GSH) is known to be present in all kinds of cells [1,2] and generally represents the major component of the low-molecu-lar-weight thiol fraction in the cell. In Escherichia coli, 25% of the sulfur is found in glutathione [ 3 ] . Relatively high concentrations (up to 10 mM) are found in yeast, liver and eye lens [4,5,6].

The main biological function of glutathione, which could justify its wide distribution, may be the protection of SH groups of proteins. It is difficult to demonstrate whether such protection would be necessary in the intact cell. The congenital disturbance in GSH biosynthesis in erythrocytes of patients with mild hemolytic anemia provides the only indication so far that the protective function of intracellular GSH is significant [7,8]. Studies of GSH levels and the regulation of glutathione synthesis are of biological interest, but in order to reveal what the intracellular occurrence of GSH means for a cell under different

2

conditions, such studies should probably be coupled with a genetic study on glutathione synthesis. We undertook both lines of investigation in E. coli K 12.

It is known that the biosynthesis of glutathione from its component ami-no acids is accomplished in two steps, each catalyzed by an individual enzyme and requiring ATP. First, a peptide bond is formed between glutamic acid and cysteine by 7-glutamylcysteine synthetase (EC 6.3.2.2). Then, the second pep-tide bond is formed between the product 7-glutamylcysteine and glycine by glutathione synthetase (EC 6.3.2.3).

glutamic acid + cysteine + ATP -^ 7-glutamylcysteine + ADP + Pj (1) 7-glutamylcysteine + glycine + ATP ->• glutathione + ADP + Pi (2)

Tlie overall synthesis of glutathione in extracts of E. coli was studied by Samuels [ 9 ] . The present study provides some information about the separate activities of the two synthetases and their regulation.

Materials and Methods

The E. coli K 12 strain AB 1157 {thr, leu, proA, his, argB, thi, strA) was used. The genetic symbols are those used by Taylor [10].

Media

The minimal medium used was that described by Vogel and Bonner [11], supplemented writh 0.2% glucose. The necessary growth factors were added in the following concentrations (/ig/ml): threonine 25, leucine 50, proline 25, arginine 50, histidine 10 and thiamine 1. Minimal medium without any supple-ments was used for washing procedures and for diluting cell suspensions.

For incorporation of ^' S into bacterial cells, minimal medium was used with MgQa instead of N^S04 and supplemented with 118 mg/1 Na2^'S04 (spec. act. 6 Ci/mol).

Preparation of extracts

Cultures grown aerobically at 37° C were harvested in the early stationary phase of growrth (unless otherwise explicitly stated) by centrifuging.

Growth was followed turbidimetrically in a Klett-Summerson colorimeter. The cells were washed once with cold minimal medium. Suspensions of 1 g wet weight cells in 5 ml minimal medium were treated with an MSE 100 Watt Ultrasonic Disintegrator for 5 min at 0° C. The sonicated cell suspensions were centrifuged to give cell-free extracts. The supernatant fractions were used in enzyme assays immediately or chilled and stored at — 20° C.

The protein concentration was determined by the method of Lowry et al. [12] with bovine serum albumin as standard.

Assays of j-glutamylcysteine synthetase

The formation of 7-glutamylcysteine on incubation at 37° C for 15 min was measured. The incubation mixture (4 ml, final pH 8.5) had the following composition: 20 /xmol ATP, 40 /zmol phosphoenolpyruvate, 0.032 mg pyruvate

3 kinase, 40 ixmol MgS04, 400 jumol KCl, 60 ixmol cysteine, 60 jumol glutamic acid, 0.4 ml 1 M diethanolamine/HCl buffer pH 9.15, 4.0 mg bovine serum albumin and 0.8 ml cell-free extract. After incubation at 37°C, 6.6 ml of ice-cold 3.2% sulphosalicylic acid was added. The suspension was allowed to stand at 0°C for 20 min and then centrifuged at 3000 X g for 10 min. The supernatant was electrolytically reduced by the method of Dohan and Wood-ward [13] and assayed for 7-glutamylcysteine colorimetrically with the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid), after removal of the cysteine by reaction with glyoxylic acid as described by Jackson [14]. Absorbances were read in a Zeiss PMQ II spectrophotometer.

The assay described above was less suitable for studying the inhibitory effects of GSH and GSSG on enzyme activity. In these experiments, the incu-bation mixture (final volume 1 ml) contained [U-' "C] glutamic acid (spec. act. 120 /.iCi/mmol) and the enzyme reaction was terminated by the addition of 0.1 ml 1 M trichloroacetic acid. Denatured protein was removed by centrifugation. To determine the 7-glutamylcysteine formed, 20-jul portions of the supernatant were subjected to electrophoresis on Whatrnzm 3MM paper strips for 16 h at 13 V/cm and 4°C in a buffer consisting of acetic acid/pyridine/water (70 : 15 : 2000), pH 3.9. Under these circumstances glutamic acid moved 7—11 cm in the direction of the positive electrode. 7-Glutamylcysteine and its oxidized form diglutamylcystine were found 20—26 cm from the origin in the same direction. The paperstrips were dried, cut into 1-cm sections and counted in a toluene-based scintillation fluid in a Nuclear-Chicago liquid scintillation counter. Assay of glutathione synthetase

The assay conditions were based on the method of Mooz and Meister [15] for crude extracts, with some modifications. The rate of formation of labeled GSH from 7-glutamylcysteine and [U-''' C] glycine was determined. 7-glutamyl-cysteine was prepared by the enzymic method of Strumeyer and Block [16]. The reaction mixture (0.5 ml, final pH 8.5) contained: 2.5 /zmol ATP, 5 /xmol phosphoenolpyruvate, 0.004 mg pyruvate kinase, 2.5 jUmol 7-glutamylcysteine, 7.5 iimol [U-''*C]glycine (spec. act. 60 iiCi/mmol), 5/imo! MgS04, 50 /imol KCl, 0.05 ml 1 M diethanolamine/HCl buffer pH 9.05, 0.5 mg bovine serum albumin and 0.1 ml cell-free extract. After incubation at 37° C for 30 min, the reaction was stopped by the addition of 0.05 ml 1 M trichloroacetic acid and the mixture then centrifuged. The supernatant was analysed by paper electro-phoresis as described above. Electroelectro-phoresis was carried out at 20° C for 120 min. GSH moved 2—3 cm towards the anode and glycine moved 0.5-1.5 cm in the opposite direction. If complete reaction mixtures were employed; the plot of activity versus enzyme concentration was linear.

Determination of glutathione level

Relative amounts of GSH were estimated by measuring the radioactivity of the trichloroacetic acid-soluble fraction of ^' S-labeled cells. 80—160 mg wet weight of ^' S-labeled cells was suspended in 4 ml 5% trichloroacetic acid. The suspension was allowed to stand at 4°C for 30 min and then centrifuged. The sediment was dried overnight at 110° C. Trichloroacetic acid was removed by shaking the supernatant with ether. Aliquots (25 ^1) of the aqueous phase were

4

pipetted on to Whatman No. 1 paper discs, dried and counted as described above.

The GSH concentration was measured enzymically by the method of Klotzsch and Bergmeyer [17]. Cell pellets, sedimented at 8000 X ^ in 20 min, were suspended in minimal medium and sonicated as described above. Approx. 5% of the GSH was converted to GSSG by the perchloric acid. The total of GSH and GSSG found by this method was used in the calculation of the GSH concentration. The method of Srivastava and Beutler |T.8] was used to estimate the "true" GSSG concentration.

Chemicals

GSH and L-amino acids were obtained from Merck; GSSG, ATP, phospho-enolpyruvate, pyruvate kinase (200 U/mg), NADPH, glyoxalase 1 and glutathi-one reductase from Boehringer; Na2^'S04, L-[U-'"C] glutamic acid and

[ U-'" C] glycine from The Radiochemical Centre, Amersham; carboxypeptidase A from Worthington Biochemical Corporation; methylglyoxal from the Sigma Chemical Co.; 5,5'-dithiobis-(2-nitrobenzoic acid) from-K and K Laboratories; thiamine hydrochloride from BDH Chemicals. / Results

pH optima

The effect of the pH on the activity of 7-glutamylcysteine synthetase and glutathione synthetase is illustrated in Fig. 1. Since the incubation mixtures contained weakly acid compounds, useful buffer action was obtained only in the upper pH range of the buffer systems. The pH/activity profiles of both enzymes were broad. Both enzymes showed maximum activity at pH 8.5. Stability

Several successive freezings and thawings of the cell-free extract had no effect on the activities of the enzymes. No reduction in the activity of

7-gluta-90 95 pH ,

Fig. I. pH/activity profiles for (A) 7-glutamylcysteme synthetase and (B) glutathione synthetase. For each curve one extract was used. Activities are expressed as Mmol peptide formed per ml of Incubation mixture in the times indicated. Buffers employed (final concentration 0.1 M) were: o o, triethanolamine/ HCl/NaOH; • •, diethanolamine/HCl; '^ ^, borate/NaOH.

5 •2 u E 5 '-2 ?. 10 _, ^ ^ 0 8 o 1-06 5, •go.« F £ 0 7 X in o B -L . ^ / - / / _ / • / - / / - / / / / , , pH8 5 1 J 1 1 . . -_ . _ 15 30 45 60

incubation time ( m m ) 30 60 90 120 150 180 incubation time(min)

Fig. 2. Effect of incubation time on peptide formation with (A) 7-glutamylcysteine synthetase and (B) glutathione synthetase. Reaction mixture compositions were as described under Materials and Methods. Incubations were carried out at pH 8.5 and 37°C.

mylcysteine synthetase was observed after storage at —20"C for two months, while glutathione synthetase lost 20% of its activity under these conditions.

Fig. 2 shows the progress curves of the enzyme-catalysed reactions. The reaction catalysed by glutathione synthetase was reasonably linear with time up to about 30 min. The rate of synthesis of 7-glutamylcysteine was less stable at early times. It can be seen further that under the incubation conditions em-ployed, the initial velocity of 7-glutamylcysteine synthesis is approx. two times higher than that of the reaction catalysed by glutathione synthetase.

Substrate saturation kinetics

Figs 3 and 4 show the substrate saturation curves of the enzymes. The II

020

-015

010

005

glutamic acid cysteine

8 10 ' 5(mM)

U 8 10 " U

S(mM)

Fig. 3. Substrate saturation kinetics of 7-glutamylcysteine synthetase. The incubation conditions were as described under Materials and Methods except for the concentr. tions indicated o n the abscissa. V is expressed as fimoi 7-glutamylcysteine formed in 1 5 min per mg of protein m the extract.

6 V 0.30 0 20 0 0 glycine

1 ^

1 ^

f ,

Fig. 4. Substrate s a t u r a t i o n kinetics of g l u t a t h i o n e s y n t h e t a s e . The i n c u b a t i o n c o n d i t i o n s were as de-scribed u n d e r Materials and M e t h o d s e x c e p t for t h e i n d i c a t e d c o n c e n t r a t i o n s on t h e abscissa. V is ex-pressed as Minol of g l u t a t h i o n e formed in 3 0 min per mg of protein in t h e e x t r a c t .

curves were of the usual hyperbolic form, except for 7-glutamylcysteine. Con-centrations of 7-glutamylcysteine higher than 2 mM had an inhibitory effect on glutathione synthetase, probably due to the oxidized form diglutamylcystine, which is present in sufficient quantity at that concentration.

Line weaver-Burk plots are given as insets in the figures. The Xm values calculated from the intercepts on the abscissa for glutamic acid, cysteine, 7-glutamylcysteine and glycine were 1.1 ' 10"', 8.0 " 10"", 6.3 • 10"" and 4.0 ' 10"" M respectively.

GSH and enzyme levels

Glutathione levels and the activities of the two synthesizing enzymes were determined at different phases of grovrth. The results shown in Fig. 5 demon-strate a marked increase in the glutathione content during the log phase and up to the beginning of the stationary phase. The enzyme levels, however, remained constant during the whole growth cycle. In this experiment the radioactivity of the trichloroacetic acid-soluble fraction of washed ' ^ S-labeled cells was taken

as the glutathione level. It has been shown by Roberts et al. [3] with E. coli B that in this fraction at least 95% of the ' ^ S is in glutathione. Our chromato-graphic analysis of the trichloroacetic acid-soluble fraction of strain AB 1157 is in good agreement with this finding. It is known that in the cells a small percentage of glutathione may exist in the oxidized form. We measured the concentrations of GSH and GSSG enzymically in cell pellets. The amounts of GSH found in mid-log-phase cells and stationary-phase cells were 1.15 mg and 2.23 mg per ml cell pellet respectively, which correspond to 3.5 mM and 6.6 mM. GSSG was undetectable in mid-log-phase cells and stationary-phase cells. Repression of enzyme synthesis

The effect of GSH and GSSG in the growth medium on the levels of both enzymes was examined.

7

8 10 time{ hours)

Fig. 5. G l u t a t h i o n e c o n t e n t and activities of (^'- '') 7-glutaniylcysteine s y n t h e t a s e and (- - c )

g l u t a t h i o n e s y n t h e t a s e as a function of g r o w t h phase. GSH c o n t e n t ( • - ») is expressed as radioactivi-ty of 2 5 jUl of t h e t r i c h l o r o a c e t i c acid-soluble fraction per mg d r y weight of cells. The e n z y m e activities are expressed as t h e a m o u n t s of 7-glutamylcysteine formed in 1 5 min and of GSH formed in 30 min p e r mg of p r o t e i n in t h e e x t r a c t s . The different g r o w t h phases at which t h e cells were harvested are i n d i c a t e d by t h e d o t t e d line, representing t h e turbidimetrically m o n i t o r e d g r o w t h curve.

The maximum deviation in activity from that found in cells grown in unsupplomented minimal medium was 14%. It was concluded from the results presented in Table I that no significant repression of the enzymes occurred in the presence of GSH or GSSG. Especially at the low concentrations of GSH (0.13 mM) and GSSG (0.08 mM), stimulation rather than inhibition was ob-served.

It was of interest to know how the concentration of GSH or GSSG out-side the cells was reflected by the intracellular concentration at the time of harvesting. Measurements of GSH concentrations revealed that the E. coli strain T A B L E I

E F F E C T O F V A R Y I N G C O N C E N T R A T I O N S O F GSH A N D GSSG IN M I N I M A L G R O W T H M E D I U M ON T H E A C T I V I T I E S O F 7 G L U T A M Y L C Y S T E I N E S Y N T H E T A S E A N D G L U T A T H I O N E S Y N T H E -TASE A N D ON T H E C O N C E N T R A T I O N O F GSH IN C E L L P E L L E T S

Cells w e r e harvested in t h e early s t a t i o n a r y phase. Specific activity is given as Mmol of p e p t i d e formed p e r mg p r o t e i n in 15 rain for 7-glutamylcysteine s y n t h e t a s e and in 3 0 m i n for g l u t a t h i o n e s y n t h e t a s e . GSH is expressed in Mmol/ml cell pellet.

A d d i t i o n to m i n i m a l growth m e d i u m Specific activity 7 - G I u t a m y l c y s t e i n e s y n t h e t a s e G l u t a t h i o n e s y n t h e t a s e GSH 0.13 m M 0.26 m M 0.40 m M 1.60 m M None 0.08 m M 0.16 m M 0.80 m M G S H G S H G S H G S H G S S G G S S G G S S G 0.26 0.22 0.21 0.25 0.23 0.26 0.21 0.23 0.30 0.28 0.27 0.2B 0.27 0.29 0.24 0.26 9.6 11.3 17.8 6.6 5.1 6.5

8

-glutamylcysteine synthetase glutathione synthetase

J 1 I I I I I—I 1 1 1 L

1 3 5 7 9 1 3 5 7 9

mM mM Fig. 6. Inhibition of 7-glutamylcysteme synthetase and glutathione synthetase by GSH or GSSG. The incubation mixtures were supplemented with GSH or GSSG to the concentrations indicated on the abscissa. Enzyme activities are expressed as percentages of the specific activity obtained with the control sample.

has the ability to accumulate larger pools of GSH when cultured in the pres-ence of GSH. GSSG remained undetectable in the cells under these growth conditions.

It is unlikely that repressed conditions for glutathione synthesis already exist in cells growing in minimal medium. No derepression was observed in mutant strains which were blocked in one of the enzymes [19].

Inhibition of enzyme activity

The activities of 7-glutamylcysteine synthetase and glutathione synthetase were measured in cell-free extracts prepared from early stationary phase cells grown in minimal medium.

Enzyme assays were carried out in the absence and in the presence of GSH or GSSG in the concentration range 1- 10 mM. The results are shown in Fig. 6. It can be seen that 7-glutamylcysteine synthetase was inhibited by GSH (50% at 5.5 mM). Glutathione synthetase was inhibited by GSSG (50% at 1.5 mM) but not by GSH.

Discussion

The activity of 7-glutamylcysteine synthetase is roughly twice that of glutathione synthetase, under the incubation conditions employed. This finding agrees with the observation of Minnich et al. [20] and Sass [21] in human erythrocytes. It suggests that the second step in the synthesis of glutathione is rate limiting, but the intermediate 7-glutamylcysteine has never been found in any type of cell. The K^^ values obtained for glutathione synthetase are lower than those for 7-glutamylcysteine synthetase, but these results cannot be ade-quately discussed without knowledge of other kinetic constants of the enzymes and endogenous pool levels of the three constituent amino acids.

9 The increase in glutathione content during growth up to the beginning of the stationary phase, without increase in the levels of the .synthesizing enzymes, suggests that the availability of the amino acids is an im~portant factor for glutathione synthesis. Moreover, it indicates that there exists a reserve capacity for glutathiohe synthesis during active growth. This was also demonstrated in erythrocytes by Minnich et al. [ 2 0 ] .

GSH was found t o inhibit 7-glutamylcysteine synthetase, while GSSG inhibited the activity of glutathione synthetase. It is very hkely that the inhibi-tion by GSH is of physielo^cal significance. The concentrainhibi-tion of 5.5 mM GSH needed in the incubation mixture to produce 50% inhibition is comparable with the intracellular level measured in stationary-phase cells (6.6 ;umol/ml of packed cells). In this connection we may note the observation of Roberts et al. [3] that glutathione synthesis from ['^ S] sulphate was greatly reduced when both [ ' ^ S] sulphate and nonradioactive glutathione (0.31 mM) were present in the medium. This cannot be due to the repression, as is shown in Table I. The control of the pathway by feedback inhibition and not by repression is consis-t e n consis-t wiconsis-th consis-the possible role of gluconsis-taconsis-thione in consis-the proconsis-tecconsis-tion of cells againsconsis-t consis-toxic agents. It would be important for cells which lose their glutathione under the conditions of a chemical challenge, to have the ability to replenish GSH imme-diately.

Acknowledgements

The authors thank Dr J.W.M. Noordermeer and Ir W.R. van Dijk for their collaboration in this investigation and Mr B.W,,Groen for his skillful technical assistance.

References

1 Jocelyn, P.C. ( 1 9 5 9 ) Symposium o n Glutathione (Crook, E.M., ed.), Vol. 1 6 , p. 4 3 , Biochem. Soc. Symp. Cambridge, U.K.

2 Knox, W.E. ( 1 9 6 0 ) in The Enzymes (Boyer, P.D., Lardy, H. and Myrback, K., eds), 2nd edn. Vol. 2, p. 2 5 3 , Academic Press, New York

3 Roberts, R.B., Abelson, P.H., Cowie, D.B., Bolton, E.T. and Britten, R.J. ( 1 9 5 5 ) Studies of Biosyn-thesis in Escherichia coli Carnegie Inst. Wash. Publ. 6 0 7 , p. 3 1 8 , Washington

4 Schroeder, E.F. and Woodward, G.E. ( 1 9 3 9 ) J. Biol. Chem. 1 2 9 , 2 8 3 5 Bhattacharya, S.K., Robson, J.S. and Stewart, C.P. ( 1 9 5 6 ) Biochem. J. 6 2 , 12 6 Harding, J.J. ( 1 9 7 0 ) Biochem. J. 1 1 7 , 957

7 Boivin, P. and Galand, C. ( 1 9 6 5 ) Nouv. Rev. Fr. He'matol. 5, 7 0 7

8 Prins, H.K., Oort, M., Loos, J. A., Ziircher, C and Beckers, T. ( 1 9 6 6 ) Blood 27, 1 4 6 ' 9 Samuels, P.J. ( 1 9 5 3 ) Biochem. J. 5 5 , 4 4 1

1 0 Taylor, A.L. ( 1 9 7 0 ) Bacteriol. Rev. 3 4 , 1 5 5

11 Vogel, H.J. and Bonner, D.M. ( 1 9 6 5 ) J. Biol. C3iem. 2 1 8 , 9 7

1 2 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and RaridaU, R.J. ( 1 9 5 1 ) J. Biol. Chem. 1 9 3 , 2 6 5 1 3 Dohan, J.S. and Woodward, G.E. ( 1 9 3 9 ) J. Biol. Chem. 1 2 9 , 3 9 3

1 4 Jackson, E.G. ( 1 9 6 9 ) Biochem. J. I l l , 309

1 5 Mooz, B.D. and Meister, A. ( 1 9 6 7 ) Biochemistry 6, 1 7 2 2 1 6 Strumeyer, D. and Block, K. ( 1 9 6 2 ) Biochem. Prep. 9, 5 2

17 Klotzsch, H. and Bergmeyer, H.U. ( 1 9 6 2 ) in Methoden der Enzymatischen Analyse (Bergmeyer, H.U., ed.), p. 3 6 3 , Verlag Chemie, Weinheim

18 Srivastava, S.K. and Beutler, E. ( 1 9 6 8 ) Anal. Biochem. 2 5 , 70

19 Apontoweil, P. and Berends, W. ( 1 9 7 5 ) Biochim. BioPhys. Acta 3 9 9 , 10—22

20 Minnich, V., Smith, M.B., Brauner, M.J. and Majerus, P.W. ( 1 9 7 1 ) J. Clin. Invest. 5 0 , 507 21 Sass, M.D. ( 1 9 6 8 ) Clin. Chim. Acta 2 2 , 207

Reprinted from

Biochimica et Biophysica Acta, 399 (1975) 10—22

© Elsevier Scientific Publishing Company, Amsterdam —Printed in The Netherlands

BBA 27690

ISOLATION AND INITIAL CHARACTERIZATION OF

GLUTATHIONE-DEFICIENT MUTANTS OF ESCHERICHIA COLI K 12

P. APONTOWEIL and W. BERENDS

Biochemical and Biophysical Laboratory, Delft University of Technology, Julianalaan 67, Delft (The Netherlands)

(Received February 21st, 1975)

Summary

The thiol-oxidizing agent "diamide" (CH3 )2 NCON=NCON(CH3 )2 was used to isolate mutants of Escherichia coli K 12 deficient in the biosynthesis of glutathione. A colony-colour technique has been developed for identification of colonies of these mutants. Four glutathione-deficient mutants were isolated. They show normal growth rates in minimal medium without GSH supplementa-tion, indicating that glutathione is not involved in essential metabolic processes. In one mutant, glutathione synthetase was enturely inactive. Three mutants were deficient in 7-glutamylcysteine synthetase; in two of them, this resulted in a complete lack of GSH. These mutants were found to be more susceptible than their parent strains to a wide range of chemical agents, but did not show a greater sensitivity to X-rays. It must be concluded that the protective role of glutathione is only significant when a chemical challenge is present.

Introduction

One way of studying the metabolic role of glutathione is to observe the effects of intracellular oxidation of GSH. Azoester (methylphenyldiazene car-boxylate) and diamide (diazenedicarboxylic acid bis-(Ar,N-dimethylamide)) are reagents introduced by Kosower et al. [1,2,3], which can rapidly oxidize GSH stoichiometrically in the cell. These reagents may produce cellular damage in addition to oxidizing GSH [4,5]. Recently Srivastava et al. [6] suggested the use of stable organic hydroperoxides, ^butyl hydroperoxide or cumene hydro-peroxide. Being substrates for glutathione peroxidase the hydroperoxides stim-ulate enzymic oxidation of erythrocyte GSH. Although more specific, these agents still cause a temporary high intracellular concentration of GSSG, which complicates the interpretation of the effects.

11 In studying the function of GSH, the use of mutant cells with a deficiency in GSH biosynthesis is to be preferred to the use of cells depleted in GSH by chemical agents. The knowledge of the consequences of a low GSH level (lower than 10% of normal) comes mainly from studies of erythrocytes lacking in one of the two synthesizing enzymes [7,8].

In this paper we report the isolation of mutants of E. coli K 12 deficient in GSH biosynthesis. The techniques used for isolation of the mutants are described and the mutant strains are biochemically characterized.

The effectiveness of intracellular glutathione in protecting the cell against foreign compounds and X-irradiation is studied by comparison of the suscepti-bility of the mutants with that of the parent strains.

Materials and Methods Bacterial strains

The E. coli K 12 strains AB 1157 and KMBL 54 were supplied by the Medical Biological Laboratory TNO, Rijswijk, the Netherlands. Strain AB 1157 has the following relevant genetic markers: F", thr, leu, proA, his, argB, thi, strA. The genetic markers of strain KMBL 54 are: F", thi, pyrF, thy, lac. Media and culture conditions

Complete nutrient broth medium contained 8 g Difco nutrient broth, 5 g Difco yeast extract and 5 g NaCl per 1 water.

The minimal medium used was that of Vogel and Bonner [9] supple-mented with 0.2% glucose. The necessary growth factors were added in the following concentrations (/xg/ml): threonine 25, leucine 50, proline 25, histi-dine 10, arginine 50, thymine 20, uracil 20 and thiamine hydrochloride 1. Minimal medium without any supplements was used for washing procedures and for diluting cell suspensions.

Cultures were grown at 37° C with shaking. Growth was measured with a Klett-Summerson colorimeter. Solid media in plates contained 1.6% Difco bacto-agar. Solid minimal medium was supplemented with 1% glucose. Cell-free extracts were prepared as described previously [ 1 0 ] . In the sedimentation-velocity experiments, DNA was labeled by growing the bacteria in minimal medium supplemented with 10 ;uCi/ml [Afe-^H] thymidine (spec. act. 5 Ci/mmol), 3 /xg/ml thymidine and 250 Mg/^il deoxyadenosine.

Induction of mutants

Mutations were induced with TV-methyl-A^'-nitro-A/^-nitroguanidine accord-ing to a technique of Van de Putte [11]. Incubation was carried out with 800 ;ug/ml of the mutagen for 30 min at 37° C. Immediately after the treatment with mutagen, cells were washed with minimal medium, divided into several portions and then allowed to grow in minimal medium for phenotypic expres-sion. No GSH was added.

Penicillin enrichment with diamide

Cultures of mutagenised bacteria were diluted 1 : 50 into 40 ml minimal medium supplemented with 0.05% MgS04 and 10% sucrose, and incubated

12

with shaking at 37° C until the early logarithmic phase. Diamide was added to a concentration of 160 /xg/ml and the cultures were shaken gently at 37° C until growth began again.

This step required approx. 90 min. Penicillin G (1000 units/ml) was then added and incubation was continued for 70 min but without shaking. Penicillin action was stopped by chilling, and the cells were harvested by centrifugation at 4°C. Pellets were resuspended in 10 ml minimal medium and treated with penicillinase for 30 min at 37° C. To obtain overnight cultures, 2 ml of these suspensions were inoculated into 20 ml of minimal medium. The above proce-dure was then repeated once.

Screening

Appropriate dilutions of enriched cultures were spread on minimal agar, yielding 100—150 colonies per plate. The plates were incubated at 37° C for 48 h and then used as master plates for replica plating. The copies were screen-ed for colonies of GSH" mutants by colouring with sodium nitroprusside. 1.5 ml of a solution of sodium nitroprusside (2% in 5% trichloroacetic acid) was pipetted on to each plate and allowed to react with the colonies for approx. 20 s. The solution was then poured off and a few drops of concentrated am-monia were appHed to the surface of the agar. Prior to the colouring procedure plates and solutions were cooled down to 0—4°C to stabilize the violet-red colour of the colonies. Colonies which were unable to colour were picked from the master plates, purified and examined for their glutathione level.

An alternative isolation procedure without enrichment was also employed. Cultures of mutagenised bacteria were plated, at suitable dilutions, directly onto minimal agar containing 25 /ng/ml of diamide. The plates were incubated at 37° C for 28 h before being examined for the first time. The location of each colony was marked. The plates were then incubated at 37° C for another 40 h, and colonies which became visible in this period were picked off and grown in complete medium at 37° C. Dilutions were replated on minimal agar with and without diamide supplementation, to verify that growth inhibition was really due to the diamide. Colonies of diamide-susceptible cells were used for the determination of the colour test and the glutathione level.

Glutathione levels

The radioactivity of the trichloroacetic acid-soluble fraction of ^' S-la-beled cells was used to determine the relative glutathione level as described previously [10]. The specific activity of the Na2 ^ ^ SO4 used in the minimal medium was 0.6—1.0 Ci/mol. The mutants were identified by descending paper chromatography of the trichloroacetic acid-soluble fraction overnight, on What-man No. 1 paper with the solvent system n-butanol/acetic acid/water ( 2 : 1 : 1 ) . Chromatograms were scanned in a Packard Radiochromatogram scanner or cut into 1-cm sections and counted in a toluene-based scintillation fluid in a Nuclear-Chicago liquid scintillation counter.

Enzyme assays

The activity of 7-glutamylcysteine synthetase was measured according to the method of Jackson [12] and glutathione synthetase activity by the method of Mooz and Meister [13] with the modifications described previously [10].

13

Determination of minimal inhibitory concentrations

An agar-dilution procedure was used to determine the minimal inhibitory concentrations in minimal agar. Solutions of the agents were mixed with mini-mal agar just before pouring the agar into the plates. The plates were prepared a few hours before inoculation. The strains to be tested were grown in minimal medium until the early stationary phase and then diluted with minimal medium to a density of 10'' cells per ml. Inoculation of the plates and end-point readings were carried out as recommended in the International Collaborative Study by Ericsson and Sherris [14].

Survival curves

Samples of cell suspensions, exposed to bactericidal compounds or to X-irradiation, were diluted in cold minimal medium and plated on complete nutrient agar. The plates were then incubated at 37° C to determine the number of surviving colony-forming cells. The data are plotted as the surviving fraction N/No, where N is the number of cells surviving at the time or dose indicated and No is the number surviving without treatment.

Irradiation

Early stationary-phase cells grown in minimal medium were suspended in ice-cold 0.05 M phosphate buffer (pH 6.8). 0.5-ml samples were exposed to X-rays in air in 5-ml glass breakers, which were vibrated at 50 Hz. X-irradiation was performed using a Machlett OEG-60 tube with a 1-mm beryllium window, The tube was operated at 50 kV and 30 mA. The dose rate was approx. 600 rad/s.

Sedimentation in alkaline sucrose gradients

Cells with ^H-labeled DNA were harvested by centrifugation, washed twice with cold minimal medium and resuspended in 0.05 M phosphate buffer (pH 6.8) to give 10' cells per ml. Spheroplasts were prepared at 0°C from irradiated and unirradiated cells according to the method of Rupp and Howard-Flanders [15]. EDTA (0.16 ml of 32 mM EDTA for 0.5 ml of cell suspension) was added immediately after irradiation.

The spheroplasts (no more than 6-10* in a 20-/il portion) were lysed in 0.1 ml of 0.5 M NaOH layered on top of a 5.2-ml 5—20% sucrose gradient (adjusted to pH 12.0 with NaOH) containing 1 M NaCl. The gradients were centrifuged in a SW 50L rotor for 120 min at 30 000 rev./min and 20° C in a Beckman/Spinco Model L2 ultracentrifuge. After centrifugation, 27 fractions of 4 drops each were collected in glass counting vials. 0.5 ml of water was added to each vial followed by 13 ml of a toluene-based scintillation fluid containing 20% Triton X-100. The radioactivity was measured in a Nuclear-Chicago liquid scintillation counter.

Chemicals

The sources of the chemicals used for enzyme assays and glutathione determinations were described in the previous paper [10]. GSH, L-amino acids, thymine, thymidine, uracil, 2'-deoxyadenosine and sodium nitroprusside were obtained from Merck; A^-methyl-AT-nitro-iV-nitrosoguanidine from Aldrich

Eu-14

rope, diamide from Calbiochem, penicillin G, streptomycm and chlorampheni-col from Mycofarm, Delft; [Me-^ H] thymidine from The Radiochemical Cen-tre, Amersham. All other chemicals were mostly the purest available from commercial sources.

Results

Isolation of mutants

Attempts to isolate GSH auxotrophic mutants based on the requirement of GSH (using standard penicillin enrichment and replica-plating technique) were not successful. We then considered the possibility that mutants defective in GSH synthesis may proliferate m minimal medium without GSH supplemen-tation. For the isolation of such mutants we used the thiol-oxidizing agent diamide. Wax et al. [16] observed that addition of diamide (cone. 2 • 10"''—6 • 10"* M) to cultures of E coli B caused a lag during which no growth occurred We assumed that cells without GSH must give a longer lag. We used diamide in the penicillin-enrichment procedure and as addition to agar plates for the

selec-30 AO 50 distance of migration (cm) .25 -C20 E ' KMBL 54 ' 129 10 20 30 AO 50 distance of migration ( cm)

Figs 1 and 2 C h r o m a t o g r a p h i c identification of glutathione-deficient m u t a n t s Trichloroacetic acid-soluble fractions were p r e p a r e d f r o m early s t a t i o n a r y - p h a s e cells, w h i c h h a d m c o r p o r a t e d [ ^ ^ S ] s u l p h a t e from t h e medium. Each chromatogram contained 2 5 Ml of the trichloroacetic acid-solubie fraction.

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tion of colonies with a delay in growth. We also developed a screening techni-que based on the idea that sodium nitroprusside in the presence of trichloro-acetic acid would be able to react with the sulhydryl groups in whole colonies to produce a violet-red colour. It was to be expected that a discrimination could be made between GSH* and GSH" colonies on the basis of their ability to colour with sodium nitroprusside, since GSH represents at least 95% of the low-molecular-weight thiols [17].

Four W-methyl-TST-nitro-A^-nitrosoguanidine-induced mutants defective in glutathione synthesis were isolated. Three mutants were obtained from strain AB 1157. Mutant strain 7 was detected by its failure to colour with sodium nitroprusside after penicillin enrichment. The two other mutant strains, 821 and 830, isolated from AB 1157 and one mutant strain 129 from KMBL 54 were scored directly as colonies with a delay in growth on minimal agar supple-mented with diamide and then tested for their ability to colour with sodium nitroprusside. Colonies of the strains 821 and 129 remained white, while those of 830 gave a pinkish colour.

All mutants were finally identified by paper chromatography of their trichloroacetic acid-soluble fraction. The chromatograms (Figs 1 and 2) show that the •* ^ S of the trichloroacetic acid-soluble fractions of the parent wild-type strains AB 1157 and KMBL 54 is concentrated in two compartments which have the characteristic movements of oxidized and reduced glutathione [Rp values of 0.14 and 0.44). The Rp value (0.20) of the major component of mutant strain 830 corresponded to that of diglutamylcysteine, the oxidized form of 7-glutamylcysteine.

Glutathione content

The amounts glutathione present in the mutants were measured enzymi-cally by the method of Klotzsch and Bergmeyer [18] in cell pellets. As also described earlier [10] the total content of GSH and GSSG found by this method was used in the calculation of the GSH concentration. The results are presented in Table I. GSH could not be detected in the strains 821 and 129. Levels of synthesising enzymes

In order to locate which step in glutathione biosynthesis was blocked in the mutants, the activities of 7-glutamylcysteine synthetase and glutathione

TABLE I

GLUTATHIONE CONTENT OF PARENT-TYPE A N D MUTANT STRAINS

Cultures in minimal medium were harvested in the early stationary phase. GSH is expressed in Mmol/ml cell pellet. N D , not detectable.

Strain GSH Percentage of parent-type

AB 1157 7 8 2 1 8 3 0 KMBL 54 129 6 . 6 2 0.07 ND 0 . 8 2 6.80 ND 1 0 0 1 0 1 2 1 0 0 0

16

TABLE II

LEVELS OF GLUTATHIONE-SYNTHESIZING ENZYMES

Assays of 7-glutamylcysteine synthetase and glutathione synthetase were performed as described under Materials and Methods. Activities are expressed as percentages of the specific activities of the parent strains. Strain Relative specific activity (%)

7-Glutamylcysteine synthetase Glutathione synthetase AB 1157 7 8 2 1 8 3 0 KMBL 54 129 1 0 0 6 4 1 0 0 1 0 0 7 100 103 91 0 100 100

synthetase were assayed. The results are summarized in Table II. The mutants 7, 821 and 129 were found to be defective in 7-glutamylcysteine synthetase activity. They will be designated by gshA mutants. Mutant strain 830 had no glutathione synthetase activity at all and will be designated by the symbol gshB.

Growth curves

Fig. 3 shows the growth curves of the mutants and their parent strains in minimal medium. The maximum total growth of the mutants 7, 821 and 830 was slightly lower than that of their parent strain AB 1157. The growth curves of KMBL 54 and its mutant 129, however, were very similar. When GSH was added to the minimal medium in various concentrations (0.06 mM—0.40 mM) no specific increase in grovi^h could be observed for the mutants with respect to the parent strains.

6 8 10

time (hours)

6 8 10 time (hours)

Fig. 3. Growth curves of glutathioneTdeficient mutants and their parent strains in minimal medium. Overnight cultures in minimal medium were diluted 1 : 40 into 20 ml of fresh medium in side-arm flasks, which were incubated at 37°C in a shaking water bath. Turbidity was measured with a Klett-Summerson colorimeter. No GSH was supplemented.

17 Susceptibility to chemical agents

It has often been suggested that glutathione may be essential for the protection of SH groups in proteins. GSH is often used to protect enzymes in cell-free systems. The grovvi;h characteristics of glutathione-deficient mutants indicate that such protection is not necessary in the cell under normal condi-tions. But it is of interest to know whether the GSH is important when the cell is exposed to a chemical challenge. An agar-dilution method was used to deter-mine the minimal inhibitory concentration of a wide range of chemical agents against two mutant strains (821 and 129) and their parent-types. Not only • typical sulfhydryl reagents but also antibiotica, chemotherapeutics, food addi-tives, pesticides and other chemicals were selected for this experiment. Most of the compounds tested were found more inhibitory against both mutant strains, than against their corresponding parent-types. These compounds are listed in TABLE HI

MINIMAL I N H I B I T O R Y C O N C E N T R A T I O N S O F V A R I O U S C H E M I C A L A G E N T S A G A I N S T TWO G L U T A T H I O N E - D E F I C I E N T M U T A N T S A N D T H E I R P A R E N T S T R A I N S

An agar-dilution m e t h o d was used as described u n d e r Materials and M e t h o d s . S o m e a g e n t s had t o b e dissolved in a m i n i m u m of a c e t o n e or e t h a n o l and t h e solutions m a d e u p to volume with water. T h e final c o n c e n t r a t i o n s of a c e t o n e and e t h a n o l had n o effect o n t h e g r o w t h of any of t h e strains. R e a d i n g s were made after i n c u b a t i o n at 37 C for 24 h.

C o m p o u n d a d d e d K3Fe(CN)6 HgClj Thallium a c e t a t e Na3As03 lodoacetic acid l o d o a c e t a m i d e p - C h l o r o m e r c u r i b e n z o a t e P h e n y l m e r c u r i n i t r a t e Captan TMTD 2,4-D AT-nitroso-Af-ethylurea 5-Bromouracil P h e n y l h y d r a z i n e 8-Hydroxyquinoline SulfanUamide

Pyridine 3-sulphonic acid Propyl 4 - h y d r o x y b e n z o a t e Nitrofurazone F o r m a l d e h y d e p - M e t h y l a m i n o p h e n o I Phenol n-Butanol Dimethylsulf o x i d e Methylglyoxal S t r e p t o m y c i n C h l o r a m p h e n i c o l Sodium d o d e c y l s u l p h a t e Minimal i n h i b i t o r y c o n c e n t r a t i o n (Mg/ml) strain AB 1 1 5 7 1 - l o " 3 5 0 0 0 . 1 5 50 5 0.2 0 . 0 8 18 80 2 5 0 l(5o > 1 0 0 0 20 10 2.4 • 10^ 30 2 0 0 20 1 8 12 2 10^ . 1.6 • 1 0 * • 5 • 1 0 * 3 5 > 2 0 0 0 10 6 l O " 8 2 1 0.4 • 1 0 * 1 2 0 0 0 . 0 5 20 1.5 0 . 0 5 0 . 0 0 8 1 0 1 0 7 5 2 5 > 1 0 0 0 1 0 3 0.5 • 1 0 ^ 1 5 76 « fr 3 0 . 8 • 10^ 0.8 • 1 0 * 2 • 1 0 * 1 0 > 2 0 0 0 1 4 1 0 * KMBL 5 4 1.5 • 3 5 0 0 10* 0 . 1 5 5 0 5 0 . 2 0 . 1 1 8 30 2 5 0 50 2 0 0 3 0 10 1.2-30 2 0 0 2 0 4 0 1 8 2 • 1.6 • 5 • 3 5 ' 5 5 6 • 103 103 1 0 * 1 0 * • 1 0 * 1 2 9 0.8 • 1 0 * 1 2 0 0 0 . 0 5 1 0 1.5 0 . 0 5 0 . 0 5 1 2 5 2 5 2 5 5 0 2 0 3 0 . 5 - 1 0 ^ 1 6 7 6 8 6 3 0.8 • 10^ 0 . 8 - 1 0 * 3 • 1 0 * 1 0 2 1 4 1 0 * Abbreviations: c a p t a n , N ( t r i c h l o r o m e t h y l t h i o ) 4 c y c l o h e x e n e l , 2 d i c a r b o x i m i d e ; T M T D , t e t r a m e t h y l -thiuramdisulfide; 2,4-D, 2 , 4 - d i c h I o r o p h e n o x y a c e t i c acid; n i t r o f u r a z o n e , 5-nitrofurfuraldehyde semicar-bazone.