Glutathione S-transferase from oxadiazon treated chickpea

@X-9422/90 %3.00+0.00 Q 1990 PergamonPress plc

Phytochemisay,Vol. 29, No. 8, pp. 2431.-2435, 1990. Printedin Great Britain.

GLUTATHIONE

S-TRANSFERASE FROM OXADIAZON CHICKPEA ABDELRAHIM A. HUNAITI*

Laboratory of Biochemistry and Molecular Biology,

Department

and

TREATED

BASSAM R. ALI

of Biological

Sciences, Yarmouk

University,

Irbid, Jordan

(Received in revised form 17 January 1990) Key Word

Index-Cicer

artetinum; Leguminoseae;

chickpea;

glutathione

S-transferase;

oxadiazon.

Abstract-Glutathione S-transferase was purified more than 150-fold with ca 70% recovery from chickpea shoots after treatment with 10 ppm of the herbicide oxadiazon. The purification steps involved ammonium sulphate precipitation, gel filtration and affinity chromatography. The M, weight of the native enzyme was 47000 as determined by gel filtration and the enzyme was separated by ion exchange chromatography into five distinct isozymes named according to their elution order from a DEAE-Sephacel column as GST-I to GST-V. Sodium dodecyl sulfate-polyacrylamide electrophoresis analysis revealed the presence of one type of subunit for GST-I and GST-II with an apparent M, of 27 000 while the other three isozymes (GST-III, GST-IV and GST-V) displayed two types of subunits with M,s 29 000 and 27 000. Antibodies raised against the purified chickpea shoot glutathione S-transferase gave a single precipitin line with both wheat and corn extracts but only partial cross-reactivity was observed with purified human placenta enzyme.

INTRODUCTION

Glutathione S-transferases (EC 2.5.1.18) are a family of multifunctional proteins catalysing the conjugation of glutathione with a wide variety of hydrophopic compounds having electrophilic centres, such as certain herbicides and insecticides [ 1,2]. The resulting conjugates are usually less toxic and more water-soluble than their precursors and thus easily eliminated from the body. Glutathione S-transferases have been detected in both animals and plants and in some plant species their activities have been shown to increase in response to herbicide and herbicide safener treatments C3-63. Moreover, the initial metabolites of several herbicides such as chloroacetanilides, atrazine and thiocarbamates in some plants have been reported to be glutathione or homoglutathione conjugates [l, 7-101. Glutathione S-transferases have been studied in plants because of their role in herbicide tolerance mechanisms and their more general role in xenobiotic metabolism [ 1, 4, 111. Glutathione S-transferases have been purified from Zea mays using a combination of different protein fractionation methods [12]. Affinity chromatography using sulphobromophthalein glutathione agarose [4] and orange A agarose [l l] have also been used. For example, Mozer et al. [4] were able to isolate a novel form of glutathione S-transferase that was induced in corn by herbicide safener treatment. However, little is known about the induction of glutathione S-transferase activity in other plant species or by other herbicides. Therefore, the present study was undertaken to purify the glutathione S-transferase from chickpea (Cicer arietinum) shoots, by a new type of affinity chromatography, after

*Author

to whom correspondence

should

be addressed.

induction of the enzyme by the widely used herbicide oxadiazon. Several biochemical and immunological properties of the chickpea enzyme were also investigated. RESULTS AND DISCUSSION

Crude extracts prepared from chickpea shoots showed very low glutathione S-transferase activity. The sp. act. in the 15,000 g supernatant was 19.5 nmol min-’ mg-’ protein. However, enzyme activity in herbicide treated shoots (10 ppm oxadiazon) ranged from 45 to 58 nmol min-’ mg-’ protein. To further characterize the glutathione S-transferase activity, a new purification scheme was adopted. Ammonium sulphate fractionation (3545%) of the crude extracts precipitated most of the glutathione S-transferase activity and the sp. act. was increased from 58 to 436 nmol min-‘mg-’ protein (Table 1). Gel filtration of the enzyme on Ultrogel AcA 54 resulted in over 20-fold purification and complete recovery of the enzymatic activity in a single symmetrical peak. Cibacron blue 3GA agarose affinity column in the presence of 0.5 M NaCl retained all the transferase activity while the bulk of the protein was eluted. The enzyme could be eluted by 50 mM Tris-HCl buffer, pH 8.3, containing 10 mM reduced glutathione. This step resulted in over 150-fold purification with ca 70% re-. covery. No further enzymatic activity was observed when the column was washed with buffer containing 1.5 M NaCl. These purification procedures resulted in an enzyme preparation with a sp. act. of 9320 nmol min-’ mg-’ protein (Table 1). Gel filtration on Sephadex G-100 column with proteins of known M,s showed that native chickpea shoot glutathione S-transferase had a M, of 47 000. Polyacrylamide disc gel electrophoresis of the purified enzyme after treatment with SDS and fi-mercaptoethanol showed two

A. A. HUNAITIand B. R. ALI

2432 Table

I. Purification

Purification step Crude extract (NHMO, 35-M% Gel filtration Cibacron blue 3GA affinity

of glutathione

S-transferase from 80 g chickpea oxadiazon herbicide for 24 hr

Total act. (nmol min)

Total protein

60 500

1056

64 640 62 280

42 500

(mg)

148.5 52.5

4.56

protein bands. The M,s of the two subunits as determined by a linear plot of log M, vs relative mobilities of standard proteins in 12% SDS gel electrophoresis were 27 000 and 29000, respectively. This observation together with the M, of the native enzyme strongly suggests that the present glutathione S-transferase has a dimeric structure. To establish the best conditions for the storage of the enzyme, aliquots of the purified enzyme were stored at different conditions. At room temperature (-2.5”) the enzyme lost ca 50 and > 90% of its activity after one and two days, respectively, while at 4” the enzyme lost ca 50% of its activity after four days. Upon daily freezing and thawing, the enzyme lost ca 25% of its activity after the first day then a gradual loss of activity was observed subsequently. Storage of the enzyme at -2oi for seven days, then thawing resulted in a ca 25% loss of the original activity. When the purified glutathione S-transferase was heated at 80” complete loss of activity was observed within 4 min, while incubation of the enzyme at 60” resulted in 50% loss of activity after 7 min. Ouchterlony double-diffusion analysis showed that rabbit antibody prepared against the purified chickpea glutathione S-transferase gave a single precipitin line when purified enzyme or crude enzyme was used as the antigen. The antibody also gave a single precipitin line with crude extracts prepared from wheat and corn with complete fusion of the lines suggesting complete identity with chickpea enzyme. On the other hand glutathione Stransferase from human placenta showed a partial immunoreaction with chickpea glutathione S-transferase antibody. The enzymatic activity was progressively inhibited when the purified enzyme (20 pg) was incubated with increasing amounts of the chickpea anti glutathione Stransferase and finally only ca 38% of the activity was inhibited. DEAE-Sephacel was used to separate the different isozymes of glutathione S-transferase obtained from oxadiazon treated chickpea. The protein obtained after ammonium sulphate fractionation chromatographed into five enzyme activity peaks, using a O-O.4 M NaCl gradient eluent. These peaks were separately pooled and named GST-I, GST-II, GST-III, GST-IV and GST-V according to their order of elution from the column (Fig. 1). The separated isozymes were applied separately to a Cibacron blue affinity column and the enzymatic

Specific act. (nmol min mgprot.) 58 436 1180

9 320

I

shoots

treated

Purification (fold)

with 10 ppm

Recovery

(o/u)

1

100

1.5 20

106 102

160

70

activity recovered. SDS-polyacrylamide gel electrophoresis of the purified isozymes, revealed the presence of only one protein band for GST-I and GST-II with an apparent M, of 27000, while the other three isozymes (GST-III, GST-IV and GST-V) displayed two protein bands with M,s of 29 000 and 27 000. Several affinity chromatography techniques have been used to purify glutathione S-transferase from various sources [4, 11, 13.-181. However, we find that some of these techniques are unapplicable to the chickpea enzyme. The use of affinity chromatography on immobilized Cibacron blue 3GA reported here for the first time for the purification of chickpea glutathione S-transferase resulted in a homogeneous enzyme preparation as judged by SDS-polyacrylamide gel electrophoresis. The purification scheme used in the present study is rapid, easy to use and gives relatively high yield of enzyme activity compared to other methods used for the purification of plant glutathione S-transferases 14, 12, 181. The presence of considerable glutathione S-transferase activity in chickpea shoots treated with oxadiazon enabled us to purify the enzyme from this plant species and to compare it with other glutathione S-transferases from other plant species [4, 12, IS]. The subunit composition of the chickpea enzyme is similar to the transferases isolated from Zea mays [4] and wheat flour [ 181. The present enzyme has a M, which is similar to that reported for Zea (50 000) [4] as well as for other mammalian glutathione S-transferases [19]. Antibodies raised against chickpea enzyme crossreacted with complete identity with the wheat and Zea enzymes but only partial cross-reactivity was observed with human placental enzyme, suggesting that the present enzyme is immunologically similar to the corn and wheat enzymes and partially related to human placenta enzyme. Multiforms of glutathione S-transferase have been isolated from several animal and plant sources [4, 201, for example, Zea contained three distinct forms [4]. Similarly, oxadiazon treated chickpea shoot contains at least five distinct forms of glutathione S-transferase, some of which are homodimers of a 27000 subunit (GST-I and GST-II) while others are heterodimers of 27000 and 29000 subunits (GST-III, GST-IV and GST-V). It has been reported that substituted S-chlorotriazine herbicides, such as atrazine and simazine, are detoxified through glutathione S-transferase in certain plants Cl]. Oxadiazon has been reported to be enzymatically trans-

Chickpea glutathione S-transferase

5b Fraction

number

Fig. 1. Separation of chickpea shoot glutathione S-transferase isozymes by chromatography of 35-65% ammonium sulphate precipitated proteins on a DEAE-Sephacel column (2.5 x 30 cm). Eltition was carried out using a (O-O.4M) NaCl gradient; fractions (5 ml) were collected. The five glutathione S-transferase activity peaks (GST I-V) were separately pooled and further purified on a Cibacron blue 3GA affinity column. Protein was determined by measuring the absorbance at 280 nm.

formed in rice to dealkylated compounds, oxidized alcohol and carboxylic acid as major metabolites [21, 221. Whether oxadiazon is transformed through glutathione S-transferase or by some other mechanisms is still unclear. However, the availability of rapid purification procedures such as the present one will contribute in part to better understanding of oxadiazon biotransformation in vivo and in vitro. EXPERIMENTAL

Chemicals. Reduced glutathione (GSH), bovine serum albumin, Cibacron 3GA agarose, polyacrylamide, NJ-methylene bisacrylamide, SDS, Sephadex G-100, DEAE-Sephacel, Coomassie brilliant blue R-250, gel filtration protein standards, SDSelectrophoresis protein standards, complete and incomplete Freund’s adjuvants were obtained from Sigma. l-Chloro-2,4dinitrobenzene (CDNB) was obtained from Aldrich. Oxadiazon was obtained from Rhone-Poulenc, Ultrogel AcA 54 from LKB. Other chemicals and reagents were of highest purity available. Enzyme assay. Glutathione S-transferase was assayed spectrophotometrically at 340 nm by measuring the rate of l-chloro-2,4dinitrobenzene conjugation as a function of time. Assay conditions described in ref. [4] were used except that the vol. was increased to 3 ml. Assay mixts contained 2920 ~1 0.1 M K-Pi buffer, pH 6.5, 50 ~1 of 0.1 M GSH, 30 ~1 of 0.1 M l-chloro-2,4-

dinitrobenzene and enzyme soln in a total vol. of 3 ml. Enzyme activity was expressed as nmol min- ’ sp. act. as nmol min- ’ mg-’ protein. Protein concn was determined by the method of ref. [23] using bovine serum albumin as standard. PVP (5% w/v) was added to minimize the possible inhibitory effect of plant phenolic compounds as previously described [24]. Purijcation of glutathione S-transferuse. Prepn of crude extract. Shoots of seven-day-old chickpea (Cicer artietinum cv UJC107) treated for 24 hr with 10 ppm oxadiazon were excised, rinsed with H,O and dried by blotting with filter paper. Shoots were pulverized in liq. N, using a Waring blender until a fine powder was obtained. All further steps were carried out at O-4” unless otherwise indicated. The resulting powder was then suspended in 1: 1 w/v 0.1 M K-Pi buffer, pH 7.0, containing 5% w/v PVP. The homogenate was centrifuged at 15000 g for 15 min. and the supematant filtered through glass wool to remove floating materials. The filtrate was the source of enzyme. (NH&SO, fractionation. Crude extracts were subjected to fractionation by addition of 20% (NH&SO, and the pptd proteins removed by centrifugation. The supernatant was further treated with 20.9% (NH&SO, and the pptd proteins dissolved in a min. vol. of 10 mM K-Pi buffer, pH 7.0, and dialysed overnight against the same buffer. The dialysed enzyme soln was centrifuged at 15 000 g for 5 min to remove denatured proteins and the clear supernatant used for the next step. Gel filtration on Ultrogel AcA 54. The enzyme soln (7 ml containing 21.1 mg

2434

A. A. HUNAITI and B. R. ALI

proteinml‘) was carefully loaded onto an Ultrogel AcA 54 column (2.5 x 80 cm) which was pre-equilibrated with 10 mM K-Pi buffer, pH 7.0. Elution was carried out with the same buffer at a flow rate of 1 mi min-‘. Frs (5 ml) were collected and assayed for glutathione S-transferase activity and for protein. Frs containing the highest enzyme activity were pooled. Chromatography on Cibacron blue 3GA agarose. Pooled frs (36 ml containing 1.46 mg protein ml-‘) were applied to a Cibacron blue 3GA agarose column (2 x 10 cm), which was pre-equilibrated with 10 mM K-Pi buffer, pH 6.0, at a flow rate of 1 ml per 3 min. The column was then washed with the same buffer until no more proteins were detected in the eluent. A second wash with 50 mM Tris-HC1 buffer, pH 8.3, containing 0.5 M NaCl, was also carried out until no further proteins appeared in the eluent. Elution of glutathione S-transferase was carried out with 100 ml 50 mM Tris-HCl buffer, pH 8.3 containing 10 mM GSH. The flow rate was kept at 1 ml per 3 min and frs (5 ml) were collected. Frs having glutathione S-transferase activity were pooled and dialysed immediately for 14 hr against 40 vols of 10 mM K-Pi buffer, pH 7.0. The dialysed enzyme soln was coned by ultrafiltration using an Amicon ultrafiltration unit fitted with a PM 10 membrane. The column was washed with 1.5 M NaCl and reequilibrated with 10 mM K-Pi buffer, pH 6.0, before the second run. The affinity column was regenerated after several runs with 8 M urea dissolved in H,O. Electrophoresis. SDS-PAGE in the presence of /J-mercaptoethanol was carried out on 0.8mm thick 12% slab gel electrophoresis essentially as described in ref. [25]. Before electrophoresis, protein samples were mixed with an equal amount of 75 mM Tris-HCI buffer pH 8.3, 5% /I-mercaptoethanoi, 2% SDS, 10% glycerol and 0.001% bromophenol blue and boiled for 4 min. Boiled samples were loaded onto the gel and electrophoresed at constant voltage (75 V) until the dye reached the resolving gel, then the voltage was increased to 150 V until the tracking dye reached the bottom of the gel. Gels were stained with 0.05% Coomassie brilliant blue R-250 dissolved in H,OHOAc-MeOH (5:3:4) and destained with H,O-HOAcMeOH (5 : 3 : 2). Subunit composition and M, determination. The subunit composition of the purified glutathione S-transferase was determined by 12% SDS-PAGE as described in ref. [25]. Protein stds used were phosphorylase b, 94oo0, albumin, 67ooO; ovalbumin, 45 Ooo, carbonic anhydrase, 3Ooo0, trypsin inhibitor, 20 100; and a-lactalbumin, 14400. A linear plot was constructed between log M, of protein stds and their relative mobilities. from which subunit M,s were estimated. The M, of the native glutathione S-transferase was determined by gel permeation chromatography on a calibrated Sephadex G-100 column (1.5 x 100 cm) which was equilibrated with 0.1 M K-Pi buffer, pH 7.0. Protein stds were: alcohol dehydrogenase, 150 Ooo; BSA, 67 Ooo, egg albumin, 45 Ooo; carbonic anhydrase, 3Ooo0, cytochrome c, 12400. A linear plot between log M, of protein stds vs (V,/ V,) of these stds was constructed and from it the native M, was estimated. Eject of storage conditions on chickpea glutathione S-transferase. Aliquots of purified enzyme (containing 1.14 mg protein ml- ’ ) were stored at room temp. (+- 25”), 4” and - 20” for 7 days. Enzyme activity was measured daily for each ahquot and for the sample at -20” the enzyme activity was determined after daily freezing and thawing for 7 days. Thermal stability of chickpea glutathione S-transferuse. Samples of purified enzyme (containing 1.14 mg protein ml-‘) were incubated at 60” and 80”. At 2 min intervals, aliquots were removed and immediately immersed in ice then assayed for ghttathione S-transferase as described above. Immunological studies. Antibody production. Antibody against

purified chickpea glutathione S-transferase was raised in a rabbit. Ca 3OOug of purified enzyme were thoroughly mixed with Freund’s complete adjuvant (1: 1) and the resulting emulsion (0.7 ml) inj. into the hind foot pads of the rabbit. Two more booster injs (100 pg protein each) with Freund’s incomplete adjuvant (1: 1) were made at two week intervals. Ten days after the second inj the rabbit was bled, the blood allowed to clot and the antiserum collected by centrifugation [24]. Double d&ion (Immunodiffusion). Ouchterlony double-diffusion was carried out on microscope slides coated with 1% Nobel agar dissolved in 100 mM barbital buffer, pH 8.5 1241. fmmunoprecipitin lines which appeared after overnight diffusion at 4”, were first washed with 0.15 M NaCl to remove nonagglutinated proteins, then the gels were dried at 37” and stained with Coommassie brilliant blue R-250 and destained with 10% HOAc soln. Immunotitration. The effect of rabbit antiserum on enzymatic activity was determined by immunotitration. Aliquots containing 20 pg of purified enzyme were incubated with varying concns of antiserum adjusted to 15 nl with control rabbit serum in a total vol. of 200 ~1. After 5 hr at 4”, the incubation mixts were centrifuged at 20009 for 10 min and enzymatic activity in supernatants assayed as described above. Separation of chickpea glutathione S-transferuse isozymes by DEAE-Sephacel chromatography. Enzyme solution (15 ml containing 18.3 mg proteinml-‘) obtained from the 3565% (NH,)SO, fractionation step were applied to a DEAE-Sephacel column (2.5 x 30 cm) which was pre-equilibrated with 10 mM K-Pi buffer, pH 7.0. The column was washed with the same buffer until no more proteins appeared in the eluent. The enzyme was eluted with 700 ml of a linear gradient of O-O.4 M NaCl made in the same buffer. Frs (5 ml) were collected and assayed for ghttathione S-transferase activity. Protein concn was measured by monitoring A at 280 nm. Peaks containing the highest glutathione S-transferase activity were separately pooled and dialysed against 10 mM K-Pi buffer, pH 7.0, for 14 hr. The sepd glutathione S-transferase activity peaks were applied separately to a Cibacron blue 3GA agarose column and enzymatic activity recovered as described above.

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Chickpea glutathione S-transferase

2435

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