Isolation and Characterization of Glutathione S-Transferase Isozymes ...

Plant Physiol. (1998) 117: 877–892

Isolation and Characterization of Glutathione S-Transferase Isozymes from Sorghum1 John W. Gronwald* and Kathryn L. Plaisance Plant Science Research Unit, Agricultural Research Service, United States Department of Agriculture (J.W.G.), and Department of Agronomy and Plant Genetics (K.L.P.), University of Minnesota, St. Paul, Minnesota 55108 biochemical, and sequence similarities (Buetler and Eaton, 1992). It is well established that mammalian GSTs play an important role in the detoxification of electrophilic xenobiotics (Mannervik and Danielson, 1988). Although endogenous substrates for mammalian GSTs have not been clearly defined, there is evidence that a-GSTs protect against oxidative stress by detoxifying reactive products generated by lipid peroxidation (Ålin et al., 1985; Ketterer and Coles, 1991; Singhal et al., 1992). In general, plant GSTs have not been as well characterized as mammalian GSTs. Plant cytosolic GSTs belong to the archaic u class of GSTs (Meyer et al., 1991; Marrs, 1996). This class, which is very heterogeneous in primary structure, also includes GSTs from microbes, insects, and mammals (Buetler and Eaton, 1992; Pemble and Taylor, 1992). Plant u-GSTs have been subdivided into three types (I, II, and III) based on amino acid sequence identity and conservation of intron:exon placement (Droog et al., 1995; Marrs, 1996). The best-characterized function of plant GSTs is their role in the detoxification of certain herbicide classes such as the chloroacetanilides, thiocarbamates, and s-triazines (Lamoureux and Rusness, 1989). Plant GSTs can be induced by biotic stimuli such as pathogen invasion and abiotic stimuli, such as herbicide safeners and heavy metals (Marrs, 1996, and refs. therein). Very little is known about endogenous substrates and functions of plant GSTs. Certain plant GSTs bind auxins as nonsubstrate ligands (Bilang et al., 1993; Zettl et al., 1994). A GST encoded by the maize bronze2 gene conjugates anthocyanin prior to transport into the vacuole via a tonoplast transporter (Marrs et al., 1995). There are also increasing reports of plant GSTs exhibiting

Two glutathione S-transferase (GST) isozymes, A1/A1 and B1/B2, were purified from etiolated, O-1,3-dioxolan-2-yl-methyl-2,2,2,trifluoro-4*-chloroacetophenone-oxime-treated sorghum (Sorghum bicolor L. Moench) shoots. GST A1/A1, a constitutively expressed homodimer, had a subunit molecular mass of 26 kD and an isoelectric point of 4.9. GST A1/A1 exhibited high activity with 1-chloro-2, 4,dinitrobenzene (CDNB) but low activity with the chloroacetanilide herbicide metolachlor. For GST A1/A1, the random, rapidequilibrium bireactant kinetic model provided a good description of the kinetic data for the substrates CDNB and glutathione (GSH). GST B1/B2 was a heterodimer with subunit molecular masses of 26 kD (designated the B1 subunit) and 28 kD (designated the B2 subunit) and a native isoelectric point of 4.8. GST B1/B2 exhibited low activity with CDNB and high activity with metolachlor as the substrate. The kinetics of GST B1/B2 activity with GSH and metolachlor fit a model describing a multisite enzyme having two binding sites with different affinities for these substrates. Both GST A1/A1 and GST B1/B2 exhibited GSH-conjugating activity with ethacrynic acid and GSH peroxidase activity with cumene hydroperoxide, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid and 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid. Both GST A1/A1 and GST B1/B2 are glycoproteins, as indicated by their binding of concanavalin A. Polyclonal antibodies raised against GST A1/A1 exhibited cross-reactivity with the B1 subunit of GST B1/B2. Comparisons of the N-terminal amino acid sequences of the GST A1, B1, and B2 subunits with other type I u-GSTs indicated a high degree of homology with the maize GST I subunit and a sugarcane GST.

GSTs (EC 2.5.1.18) are dimeric enzymes found in mammals, insects, plants, and microbes that catalyze nucleophilic attack by the thiolate anion of GSH at electrophilic centers of hydrophobic molecules (Mannervik and Danielson, 1988). In addition to catalyzing GSH conjugation, GSTs also exhibit GSH peroxidase activity and ligandbinding functions (Mannervik and Danielson, 1988; Marrs, 1996). Mammalian GSTs compose a multigene family; in rat liver at least 13 different cytosolic GST subunits are found as either heterodimers or homodimers (Ketterer and Coles, 1991). Mammalian cytosolic GSTs have been divided into four classes (a, m, p, and u) based on immunological,

Abbreviations: alachlor, 2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide; atrazine, 6-chloro-N-ethyl-N9-(1-methylethyl)-1,3,5-triazine-2,4-diamine); CDNB, 1-chloro-2,4,dinitrobenzene; ConA, concanavalin A; DCNB, 1,2,-dichloro-4-nitrobenzene; EPTC, S-ethyldipropylcarbamothioate; EPTC-sulfoxide, sulfoxide derivative of EPTC; fluxofenim, (O-1,3-dioxolan-2-yl-methyl2,2,2,-trifluoro-49-chloroacetophenone-oxime); ethacrynic acid, 2,3dichloro-4(2-methylene-butyryl)phenoxyacetic acid; FPLC, fastprotein liquid chromatography; GST, glutathione S-transferase; GST(CDNB), GST activity measured with CDNB as the substrate; GST(metolachlor), GST activity measured with metolachlor as the substrate; 4HNE, 4-hydroxynonenal; 9 c,t-HPO, 9-hydroperoxytrans-10,cis-12-octadecadienoic acid; 13 c,t-HPO, 13-hydroperoxycis-9,trans-11-octadecadienoic acid; I50, inhibitor concentration producing 50% inhibition of enzyme activity; metolachlor, 2-chloro-N(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide; PAS, periodic acid/Schiff.

1

This work was a cooperative investigation of the U.S. Department of Agriculture, Agricultural Research Service, and the Minnesota Agricultural Experiment Station. This is Minnesota Agricultural Experiment Station publication no. 97-1-13-0017. * Corresponding author; e-mail [email protected]; fax 1– 612– 649 –5058. 877

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GSH peroxidase activity (Williamson and Beverley, 1987, 1988; Bartling et al., 1993; Zettl et al., 1994; Flury et al., 1996), which suggests a role in protection against oxidative stress. Our previous investigations of sorghum (Sorghum bicolor) GSTs were conducted with relatively crude enzyme fractions (Gronwald et al., 1987; Dean et al., 1990). The objectives of this study were to develop a protocol to purify GSTs from etiolated sorghum shoots and to characterize isozymes that were purified to homogeneity. The protocol we developed yielded two purified isozymes: GST A1/A1, a constitutively expressed homodimer, and GST B1/B2, a heterodimer induced by the herbicide safener fluxofenim. Kinetic analysis revealed that GST A1/A1 was best described by a random, rapid-equilibrium bireactant kinetic (Bi-Bi) mechanism, whereas GST B1/B2 was best described by a multisite model that generated kinetic constants for each subunit. We also provide the first evidence to our knowledge that plant GSTs are glycosylated.

MATERIALS AND METHODS Chemicals2 [U-14C]Metolachlor, specific activity 71.5 mCi mg21, [U-14C]alachlor, specific activity 23.6 mCi mg21, and [U-14C]atrazine, specific activity, 19.0 mCi mg21, were provided by Novartis (Greensboro, NC). N-(1-14C)Propyl EPTC, specific activity, 35 mCi mmol21, was provided by ICI Americas (now Zeneca Agricultural Products, Wilmington, DE). [14C]EPTC-sulfoxide was prepared and purified as described by Casida et al. (1975). GSH and chlorotriphenyltin were obtained from Aldrich. Polybuffer 74, Sephacryl S200, and epoxy-activated Sepharose 6B were purchased from Pharmacia. Jack bean a-mannosidase was purchased from Boehringer Mannheim. All other chemicals were obtained from Sigma. S-hexyl-GSH and S-hexyl-GSH-linked Sepharose 6B were synthesized as described by Mannervik and Guthenberg (1981). 4-Hydroxynonenal-diethylacetal, provided by Dr. H. Esterbauer (Institut fu¨r Biochemie, Universitat Granz, Granz, Austria), was converted to 4HNE by acid saponification (1 mm HCl, 1 h) prior to use. 9 c,t-HPO was synthesized as described by Matthew et al. (1977) and 13 c,t-HPO was synthesized as described by Hamberg and Samuelsson (1967). Linoleic hydroperoxide was separated from the free acid as described by Matthew et al. (1977) with modifications. The extracted reaction mixture was evaporated under a vacuum, reconstituted in hexane:ether (98:2, v/v), and then applied to a silica gel column (1.5 3 26 cm) equilibrated in the same solvent. The column was washed with hexane:ether (98:2, v/v), and the hydroperoxide and unreacted linoleic acid were separated using a step gradient of increasing ether concentrations (five incre2 Product names are necessary to report factually on available data; however, the U.S. Department of Agriculture neither guarantees nor warrants the standard of the product, and the use of the name by the U.S. Department of Agriculture implies no approval of the product to the exclusion of others that may also be suitable.

Plant Physiol. Vol. 117, 1998

mental increases of 20% over 30 mL) in hexane. Fractions were monitored at 234 nm. Hydroperoxides were identified by iodometric determination (Kokatnur and Jelling, 1941) and by their unique UV spectra.

Plant Material Untreated and fluxofenim-treated (0.4 g kg21 seed) sorghum (Sorghum bicolor L. Moench var DK41Y) seeds were provided by Novartis. Forty grams of untreated or fluxofenim-treated seeds was planted 2.5 cm deep in a plastic tray (50 3 30 3 6 cm) containing 5 L of vermiculite. Each tray of seeds was watered with 4 L of deionized water and allowed to drain. Trays were covered with aluminum foil and incubated for 72 h in the dark at 30°C and 70% RH.

GST Dimer and Monomer Purification Twenty-five to forty grams of 2.0-cm apical sections from 72-h-old etiolated sorghum shoots was excised and frozen in liquid N2. The tissue was ground to a fine powder using a mortar and pestle. A premixed (30 min, 4°C) extraction buffer (0.2 m Tris-HCl, pH 7.8, 1.0 mm EDTA, 5.0 mm 2-mercaptoethanol, and polyvinylpolypyrrolidone [5%, w/v]) was added to the powder (5 mL g21 tissue), and the slurry was ground with mortar and pestle. All additional purification steps were conducted at 4°C. The homogenate was sequentially filtered through two layers each of cheesecloth and Miracloth (Calbiochem) and centrifuged (23,500g, 20 min). The resulting supernatant was concentrated by ultrafiltration (Amicon, Beverly, MA) to approximately 20 mL using a PM30 membrane and applied to an S200 HR gel-filtration column (2.5 3 49 cm) equilibrated with buffer A (20 mm Tris-HCl, pH 7.8, 1 mm EDTA, and 5 mm 2-mercaptoethanol). Active fractions were pooled and applied directly to a S-hexyl-GSH-linked Sepharose 6B column (1.25 3 49 cm) equilibrated in buffer A. The column was washed using a 300-mL linear gradient of 0 to 0.2 m NaCl in buffer A, and GST isozymes were eluted with a 300-mL linear gradient of 0 to 5 mm S-hexylGSH in 0.2 m NaCl at a flow rate of 1 mL min21. GST isozymes, which eluted as a single peak at approximately 1.5 mm S-hexyl-GSH, were quickly dialyzed and concentrated using a Centricell 20 (30,000 Mr cutoff, Polysciences, Warrington, PA). The sample was applied to an FPLC anion-exchange column (Mono-Q HR 5/5, Pharmacia) equilibrated in buffer A. The isozymes were separated using a linear gradient of 0 to 0.2 m NaCl in 40 mL followed by isocratic elution (10 mL). Fractions (0.5 mL) were collected at a flow rate of 0.3 mL min21. The peak of GST(CDNB) activity that eluted at approximately 50 mm NaCl (Fig. 1, A and B, peak 2) was applied to a chromatofocusing column (Mono-P HR 5/20, Pharmacia) equilibrated in 0.025 m piperazine-iminodiacetic acid, pH 6.3, and eluted with 35 mL of 10% polybuffer 74-iminodiacetic acid, pH 4.5. GST isozymes were concentrated and dialyzed in buffer A using a Centricon 10 (Amicon). All elution profiles are representative of experiments repeated multiple times.

Sorghum Glutathione S-Transferase Isozymes The subunits of GST A1/A1 and GST B1/B2 were purified using a 25-cm, 300-Å C18 reverse-phase HPLC column (Vydac, Hesperia, CA), as described by Ostlund Farrants et al. (1987). The solvents were water (solvent A) and 0.1% (v/v) trifluoroacetic acid in acetonitrile (solvent B). Purified GST A1/A1 or GST B1/B2 was injected onto the column at 35% solvent B, and a linear gradient (35–55% solvent B over 60 min) with a flow rate of 1.5 mL min21 was used to resolve GST proteins. Protein was detected at 220 nm. Enzyme Assays GST(CDNB) and GST(DCNB) activities were determined spectrophotometrically as described by Habig et al. (1974) with modifications. For GST(CDNB) assays, the reaction medium contained 0.1 m potassium phosphate buffer pH 7.5 or 6.5, 1.0 mm GSH, 1.0 mm CDNB, 1% absolute ethanol, and protein in a total volume of 1.0 mL. For GST(DCNB) assays, the reaction medium contained 0.1 m potassium phosphate buffer, pH 7.5, 5.0 or 1.0 mm GSH, 1.0 mm DCNB, 1% absolute ethanol, and protein in a total volume of 1.0 mL. The reaction, conducted at 25°C, was initiated by the addition of CDNB or DCNB, and the change in A340 or A345, respectively, was monitored for 120 s with a spectrophotometer (model DU-65, Beckman). All initial rates were corrected for the background nonenzymatic reaction. One unit of activity is defined as the formation of 1 mmol product min21 at 25°C (extinction coefficient at 340 nm 5 9.6 mm21 cm21 for CDNB; extinction coefficient at 345 nm 5 8.5 mm21 cm21 for DCNB). GST (metolachlor, alachlor, EPTC-sulfoxide, and atrazine) activities were determined by measuring the amount of herbicide conjugate formed as previously described (Gronwald et al., 1987; Dean et al., 1991) with modifications. All assays contained 0.1 m potassium phosphate buffer (pH 7.5 for metolachlor assays, pH 7.0 for alachlor assays, pH 6.8 for EPTC-sulfoxide assays, and pH 6.5 for atrazine assays), 1.0 mm GSH, 5.0 mm [14C]herbicide (specific activity for metolachlor and alachlor, 5 mCi mmol21; for EPTC-sulfoxide, 2 mCi mmol21; and for atrazine, 4.5 mCi mmol21), 2% absolute ethanol, and protein in a final volume of 0.5 mL. Assays were initiated by the addition of radiolabeled herbicide and incubated for 1 h at 25°C. Reactions were terminated by the addition of 0.05 mL of 55% TCA and/or 0.75 mL of methylene chloride. The nonconjugated herbicide was partitioned into the organic phase by vigorous shaking for 2 min followed by centrifugation (10,000g, 5 min). A 100-mL aliquot from the aqueous phase was added to 5 mL of Ecolume (ICN) and radioactivity was counted using liquid scintillation spectroscopy. GSH conjugates of the herbicides were identified by TLC with authentic standards. The enzymatic rate of conjugation was corrected for the background nonenzymatic rate. For metolachlor, alachlor, atrazine, and EPTCsulfoxide, 1 unit of activity is defined as the formation of 1 nmol conjugate h21 at 25°C. Enzyme assays with ethacrynic acid were performed as described by Habig and Jakoby (1981) with the exception that the GSH concentration was increased from 0.25 to 1.0

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mm in some experiments. GST assays for 4HNE were conducted as described by Ålin et al. (1985) except that the pH was increased from 6.5 to 7.5 and the GSH concentration was increased to 1.0 mm in some experiments. GST activity with cumene hydroperoxide, 9 c,t-HPO, and 13 c,t-HPO was determined using a coupled assay that measures production of GSSG (Awasthi et al., 1975). The assay medium contained 0.1 m potassium phosphate buffer, pH 7.0, 0.2 mm NADPH, 4.0 mm GSH, 1 unit of GSH reductase (type III, Sigma), 0.1 mm hydroperoxide, and 0.02 mL (1.5–3.0 mg) of purified GST isozyme in 1.0 mL. The reaction was started by the addition of hydroperoxide. All assays were conducted at 30°C (except those with ethacrynic acid, which were conducted at 25°C). Kinetic Analysis For GST A1/A1 kinetic studies, initial velocities were determined at pH 7.5 using the spectrophotometric assay described above. GSH concentrations were varied from 20 to 240 mm at fixed concentrations of CDNB that varied from 0.5 to 2.0 mm. Assay volume and ethanol concentration remained constant. Initial velocities for GST B1/B2 were determined using the standard metolachlor assay conditions described above. GSH concentrations were varied from 20 to 1280 mm at a fixed metolachlor concentration of 640 mm. Metolachlor concentrations were varied from 5 to 640 mm at a fixed GSH concentration of 1280 mm. Assay volume and ethanol concentration were kept constant. Data analysis involved determining the fit of initialvelocity data to two models: the random, rapidequilibrium Bi-Bi model (Eq. 1) and the random, steadystate Bi-Bi model (Eq. 2). Vmax@A#@B# aKAKB 1 aKA@B# 1 aKB@A# 1 @A#@B#

(1)

V1@A#@B# 1 V2@A#2@B# 1 V3@A#@B#2 K1 1 K2@A# 1 K3@B# 1 @A#@B# 1 K4@A#2 1 K5@B#2 1 K6@A#2@B# 1 K7@A#@B#2

(2)

v 5 v 5

where v is the initial velocity; [A] is the concentration of one substrate and [B] is the concentration of the other substrate; KA and KB are dissociation constants with the free enzyme for substrates A and B, respectively; a is the parameter describing the influence of the binding of one substrate on the binding of the second; and V1, V2, and V3 and K1 through K7 are combined velocity and rate constants, respectively. Nomenclature and definitions are those of Segel (1975). Kinetic constants were determined using the Grafit 3.0 computer program (Erithicus Software, Staines, UK). Goodness of fit of initial-velocity data to bireactant kinetic models was evaluated using the criteria described by Mannervik (1996). For GST B1/B2, the initial-velocity data were graphed using Eadie-Hofstee plots, and initial estimates of kinetic constants for high- and low-affinity sites were made. In initial estimates of kinetic constants, GSH and metolachlor concentrations of less than 60 and 20 mm, respectively, were used to generate constants for the high-affinity site, and

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concentrations greater than 320 and 80 mm, respectively, were used for determining kinetic constants for the lowaffinity site. Linear regression was used to fit the data in the defined concentration ranges to a straight line and kinetic constants (Km and Vmax) were determined from the axial intercepts. To correct for the contribution of the high-affinity site at high substrate concentrations and of the low-affinity site at low substrate concentrations, the method of successive corrections as described by Spears et al. (1971) was used. For the cycles of correction, GSH concentrations below 100 mm were used for the high-affinity site and those above 100 mm for the low-affinity site. For metolachlor, substrate concentrations of less than 50 mm were used for the high-affinity site and those greater than 50 mm were used for the lowaffinity site. The initial estimates of Km and Vmax for the high-affinity site were used to calculate the velocity of the high-affinity site at high substrate concentrations. The calculated velocities were subtracted from the observed velocities in the high substrate concentration range. From these corrected velocities in the high concentration range, a regression line of v and v/[S] was calculated and values for Vmax and Km for the low-affinity site were derived. These kinetic parameters were then used to calculate the velocity contribution by the low-affinity site in the low concentration range. New values for Vmax and Km for the highaffinity site were calculated from the corrected velocities as described above. Three cycles of successive corrections were performed for each substrate to obtain the corrected kinetic constants for the low- and high-affinity sites on GST B1/B2. Using the corrected kinetic constants, the predicted velocity of GST(metolachlor) activity for GST B1/B2 was calculated over the substrate concentration ranges examined using Equation 3: Vpredicted 5 V1 1 V2 5

@S#Vmax2 @S#Vmax1 1 Km1 1 @S# Km2 1 @S#

(3)

where V1, Vmax1, and Km1 are kinetic parameters for the corrected low-affinity site, and V2, Vmax2, and Km2 are the corrected kinetic parameters for the high-affinity site. Native Molecular Mass Native molecular mass of the GST isozymes was determined using gel-filtration chromatography. Purified GST A1/A1 or GST B1/B2 was applied to a Superose 12 column (Pharmacia) in buffer A (described above) containing 0.1 m KCl. The Superose 12 column was calibrated with BSA (66 kD), ovalbumin (45 kD), carbonic anhydrase (29 kD), and lysozyme (14 kD) in the same buffer. Apparent molecular mass of the enzyme was determined by interpolation of linear plots of log Mr versus RF. Gel Electrophoresis Molecular mass of GST subunits was determined by SDS-PAGE on 8% to 25% Phast gels (Pharmacia). Native pI was determined using Phast gels (IEF 4–6.5). Gels were silver stained using the protocol of the manufacturer.


Determination of I50 Values I50 values were determined for GST A1/A1 using CDNB and GSH as the substrates, and for GST B1/B2 using metolachlor and GSH as the substrates. Standard assay conditions for these substrates were as described above. I50 values were obtained by nonlinear regression analysis of the appropriate data by using the Grafit 3.0 computer program.

a-Mannosidase Treatment The conditions for the digestion of GST A1/A1 and GST B1/B2 with a-mannosidase were adapted from those of Haselbeck and Ho¨sel (1988). Purified GST A1/A1 (20 mg) and GST B1/B2 (10 mg) were denatured by boiling for 2 min in buffer B (50 mm sodium citrate, pH 4.8, and 3 mm MgCl2) containing 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol. SDS was then diluted 5-fold by the addition of buffer B containing 0.5% (w/v) octylglucoside and 0.1 mm PMSF, and the protein was boiled again for 2 min. The reaction mixture was cooled to room temperature and 0.2 unit of a-mannosidase was added. After incubation overnight at 37°C, the reaction was dialyzed against buffer B and concentrated (Centricon 10, Amicon).

Detection of Glycosylation HPLC-purified GST subunits A1, B1, and B2 and native GST A1/A1 and GST B1/B2 (with or without pretreatment with a-mannosidase as described above) were subjected to SDS-PAGE on 20% Phast gels. Protein was transferred to Immobilon P membranes (Millipore) as described by Braun and Abraham (1989) using 10 mm CAPS (3cyclohexylamino-1-propanesulfonic acid), pH 11.0, and 50 mm NaCl as the transfer buffer. Blots were blocked overnight with buffer C (500 mm NaCl, 80 mm Tris-HCl, pH 7.6, and 0.1% Tween 20) and then incubated for 1 h in buffer C containing 5 mg mL21 ConA-biotin. After two 10-min washes in buffer D (0.05% Tween 20, 137 mm NaCl, 3 mm KCl, and 25 mm Tris-HCl, pH 7.4), blots were incubated for 1 h with a 1:5000 dilution of avidin-alkaline phosphatase in buffer C. Blots were again washed with buffer D, and alkaline phosphatase activity was detected by the addition of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium, as described by Blake et al. (1984).

Polyclonal Antibody Production Purified GST A1/A1 protein (50 mg in 100 mL of PBS) and complete Freund’s adjuvant were injected as a 1:1 emulsion into the upper wing muscles of a hen. A 25-mg booster injection using incomplete Freund’s adjuvant was administered 4 weeks after the initial injection. Eggs were collected 7 to 10 d after injections and chicken IgG was purified from egg yolks as described by Jensenius et al. (1981). Titer was determined by antibody capture immunoassay (Harlow and Lane, 1988) using purified GST A1/A1.

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Antibody Cross-Reactivity

Protein Determination

GST A1/A1 and B1/B2 subunits purified by reversephase HPLC as described above were subjected to SDSPAGE using 20% homogenous Phast gels. Protein was transferred to Immobilon P membranes as described by Braun and Abraham (1989) using 10 mm CAPS, pH 11.0, and 50 mm NaCl as the transfer buffer. Blots were blocked overnight with buffer C described above. Blots were then incubated for 1 h with 6 mg mL21 purified GST A1/A1 antibody in buffer C. After two 10-min washes in buffer D (described above), blots were incubated with a 1:15,000 dilution of anti-chicken IgG-alkaline phosphatase in buffer C for 1 h. The blots were again washed twice for 10 min with buffer D. Alkaline phosphatase activity was detected by the addition of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium as described by Blake et al. (1984).

Protein was determined using the Bio-Rad assay (Bradford, 1976) with d-globulin as the standard.

N-Terminal Sequencing Purified GST A1, B1, and B2 subunits were obtained by reverse-phase HPLC as described above. HPLC fractions containing the subunits from multiple runs were pooled and the volume was reduced to 50 mL using a vacuum concentrator (Savant, Farmingdale, NY). Sequence was obtained by automated Edman degradation (model 430B Sequenator, Applied Biosystems) at the Microchemical Facility of the Institute of Human Genetics (University of Minnesota, Minneapolis).

RESULTS Purification of GST A1/A1 and B1/B2 For both untreated and fluxofenim-treated etiolated sorghum shoots, gel filtration and S-hexyl-GSH affinity chromatography yielded a fraction containing purified GST proteins in the expected range of 25 to 28 kD (data not shown). The purified GST fraction from both untreated and safener-treated sorghum shoots was applied to an FPLC (Mono-Q) anion-exchange column and eluted with a salt gradient. Fractions were collected and assayed for GST(CDNB) and GST(metolachlor) activity. In untreated shoots (Fig. 1A), there were two peaks exhibiting GST activity with CDNB but little or no activity with metolachlor. The first peak (peak 1) did not adhere to the column and was collected in the flow-through. The second peak (peak 2) bound to the Mono-Q column and eluted at approximately 50 mm NaCl. Fluxofenim-treatment induced six GST peaks (peaks 3 through 8) that exhibited activity with metolachlor but little or no activity with CDNB (Fig. 1B). The constitutively expressed peaks 1 and 2 were also present in fluxofenim-treated shoots. Although there was some variability between isolations, in most cases safener treatment increased the GST(CDNB) activity of peak 2. A

Figure 1. FPLC anion-exchange chromatography of S-hexyl-GSH affinity-purified GST protein from untreated (A) and fluxofenim-treated (B) sorghum shoots. Fractions were assayed for GST(CDNB) activity (E) at pH 7.5 and for GST(metolachlor) activity (F) as described in “Materials and Methods.”

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trend toward less induction of GST(CDNB) activity of peak 2 relative to peak 1 was observed with aging of fluxofenimtreated seeds (data not shown). The lack of binding by peak 1 (Fig. 1) was not due to column overloading, since the peak was not retained when reapplied to the column. Reapplying peak 2 to the column resulted in elution at the original salt concentration and did not lead to the appearance of the nonbinding peak 1. It was considered that the lack of binding of GSTs in peak 1 may have been due to retention of S-hexyl-GSH, which was used to elute GST protein from the S-hexyl-GSH affinity column. However, extensive dialysis of GST protein in peak 1 to remove any bound S-hexyl-GSH did not result in binding of peak 1 protein to the column. The Mono-Q elution profiles for GST(CDNB) and GST(metolachlor) activity in untreated and fluxofenim-treated sorghum shoots (Fig. 1) were similar but not the same as those previously reported by Dean et al. (1990). The profiles were similar in that they showed that fluxofenim (formerly CGA-133205) treatment induces multiple GST isozymes that exhibit relatively high activity with a herbicidal concentration of metolachlor (5 mm). However, the Mono-Q elution profiles shown in Figure 1 differ from those of Dean et al. (1990) in the number of peaks of GST activity and the salt concentrations at which they eluted. These differences may be due to the fact that Dean et al. (1990) used a different sorghum variety and Mono-Q chromatography was performed with a crude, desalted extract instead of with purified GST protein. Although SDS-PAGE of peak 2 (Fig. 1) indicated a single band at 26 kD, native IEF gel electrophoresis indicated the

Figure 2. Chromatofocusing of peak 2 (Fig. 1) from untreated (A) and fluxofenim-treated (B) sorghum shoots. Fractions were assayed for GST(CDNB) (E), GST(metolachlor) (F), and GST(alachlor) (Œ) activity as described in “Materials and Methods.” GST(CDNB) activity is expressed as micromoles per minute per milliliter; GST(metolachlor) and GST(alachlor) activities are expressed as nanomoles per hour per milliliter.


presence of three isozymes in this fraction (data not shown). FPLC Mono-P chromatofocusing was used to resolve the constitutively expressed isozymes in peak 2. For both untreated and safener-treated shoots, three peaks (2a, 2b, and 2c) eluted at approximately the same pI values, 4.95 to 5.00, 4.75 to 4.90, and 4.55 to 4.60, respectively (Fig. 2). The three peaks did not elute in the same fractions because of differences in the amount of protein in the peak 2 fraction from untreated and safener-treated shoots. Of the three peaks, safener induction was evident for peak 2c, which exhibited a high level of activity with CDNB, low activity with alachlor, and little or no activity with metolachlor. Although, as mentioned above, there was variability in the relative induction of GST(CDNB) activity of peak 2 compared with peak 1 in fluxofenim-treated seeds (Fig. 1), an increase in GST(CDNB) activity of peak 2c (Fig. 2B) was always observed in safener-treated shoots. Preliminary experiments using SDS-PAGE and native IEF indicated that most peaks in the Mono-Q (Fig. 1B) and Mono-P (Fig. 2B) elution profile of fluxofenim-treated sorghum shoots contained multiple GST isozymes. For example, peak 5 (Fig. 1B) contained at least four native isozymes (data not shown). However, it was determined that peak 2c (Fig. 2B) and peak 6 (Fig. 1B) in safener-treated shoots contained single isozymes that were designated GST A1/A1 and GST B1/B2, respectively. As indicated by SDSPAGE, GST A1/A1 is a homodimer with a subunit molecular mass of 26 kD, whereas GST B1/B2 is a heterodimer composed of 26-kD (B1) and 28-kD (B2) subunits in equal proportions (Fig. 3).


Figure 3. SDS-PAGE of GST A1/A1 (Fig. 2B, peak 2c) and GST B1/B2 (Fig. 1B, peak 6). Lanes 1 and 4, Molecular mass standards; lane 2, 100 ng of purified GST A1/A1; lane 3, 100 ng of purified GST B1/B2. Protein was visualized by silver staining.

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Figure 4. Native IEF gels of GST A1/A1 (Fig. 2B, peak 2c) and GST B1/B2 (Fig. 1B, peak 6). A, Lane 1, IEF standards; lane 2, 300 ng of purified GST A1/A1. B, Lane 1, IEF standards; lane 2, 300 ng of purified GST B1/B2. Protein was visualized by silver staining.

That both GST A1/A1 and GST B1/B2 represented single isozymes was confirmed by native IEF gels, which showed the presence of a single band (Fig. 4). Native IEF gels of GST A1/A1 and GST B1/B2 also indicated that both isozymes are acidic proteins with pI values of 4.9 and 4.8, respectively. Native Mr values for GST A1/A1 and GST B1/B2 determined by gel filtration were 43,000 and 50,000, respectively, indicating that both isozymes are dimers. The native Mr for GST B1/B2 is close to the calculated value of 54,000. However, the native Mr for GST A1/A1 is significantly less than the calculated value of 52,000. These results indicate that GST A1/A1 and GST B1/B2 do not share a similar topography. Purification tables for GST A1/A1 and GST B1/B2 are provided in Tables I and II, respectively. S-hexyl-GSH affinity-purified GST protein represented approximately 1.3% of total soluble protein in crude extracts from safenertreated, etiolated shoots. This compares favorably with GST proteins composing 1 to 2% of the total soluble protein in etiolated maize shoots (Mozer et al., 1983). An important step in the purification was the S-hexyl-GSH affinity column. This column bound greater than 90% of the applied GST(CDNB) and GST(metolachlor) activity. Although the use of the column resulted in a large increase in specific activity, there was a significant reduction in yield. Previous investigations using S-hexyl-GSH affinity columns to purify plant GSTs have indicated that yield is sacrificed for purity (Williamson and Beverley, 1988; Irzyk and Fuerst, 1993). It should be noted that the fold purification of GST A1/A1 and GST B1/B2 indicated in Tables I and II is underestimated,

since the crude fractions from sorghum shoots contained several isozymes active with the substrates (CDNB or metolachlor) assayed during purification. Characterization of GST A1/A1 and GST B1/B2

Substrate Specificity A comparison of the substrate specificities of GST A1/A1 and GST B1/B2 is presented in Table III. Two assay conditions were used for the model substrates CDNB and DCNB. The first condition was that developed by Habig et al. (1974) for assaying rat liver GSTs; the second involved a change in either pH or substrate concentration that resulted in higher activity. For GST A1/A1, GST(CDNB) activity was higher when assayed at pH 7.5 than at pH 6.5. For DCNB, activity was higher when the GSH concentration was reduced from 5 to 1 mm. These results indicate that standard assay conditions used to measure the activity of mammalian GSTs for the model substrates CDNB and DCNB may not be optimal for plant GSTs. GST A1/A1 exhibited relatively high activity with CDNB but low activity with DCNB. Compared with GST A1/A1, GST B1/B2 exhibited lower activity with CDNB and no activity with DCNB. GST A1/A1 exhibited relatively low activity with the chloroacetanilide herbicides metolachlor and alachlor but

Table I. Purification of GST A1/A1 (Fig. 2B, peak 2c) from fluxofenim-treated sorghum shoots Fraction

Total Protein

mg

Crude extract Gel filtration (S200) Affinity (S-hexyl-GSH) Anion-exchange (Mono-Q) Chromatofocusing (Mono-P) a

203 99 2.6 0.4 0.016

Total Units

mmol min

154 159 88 24 1.3

21

Specific Activity

mmol min

21

0.76 1.61 34 54 81

Yield and purification values are based on total GST(CDNB) activity in crude extracts.

mg

21

Yielda

Purificationa

%

-fold

100 103 57 16 0.8

1 2 45 71 107

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Table II. Purification of GST B1/B2 (Fig. 1B, peak 6) from fluxofenim-treated sorghum shoots Fraction

Total Protein

mg

Crude extract Gel filtration (S200) Affinity (S-hexyl-GSH) Anion-exchange (Mono-Q) a

Total Units

nmol h

203 99 2.6 0.0075

Specific Activity

21

nmol h

278 244 54 1.9

21

mg

21

1.37 2.46 21 260

Yielda

Purificationa

%

-fold

100 88 19 0.7

1 2 15 190

Yield and purification values are based on total GST(metolachlor) activity in crude extracts.

was selective for alachlor over metolachlor. In contrast, GST B1/B2 exhibited relatively high activity with both metolachlor and alachlor. Both GST A1/A1 and GST B1/B2 exhibited low activity with atrazine as a substrate. GST A1/A1 displayed no activity with EPTC-sulfoxide, whereas GST B1/B2 exhibited activity with this substrate. GSH peroxidase and GSH-conjugating activities of GST A1/A1 and GST B1/B2 with products of lipid peroxidation were also examined (Table III). Cumene hydroperoxide is a model substrate for determining GSH peroxidase activity with organic hydroperoxide substrates (Mannervik and Danielson, 1988). Both GST A1/A1 and GST B1/B2 exhibited GSH peroxidase activity with this substrate. For both isozymes, GSH peroxidase activity with 9 c,t-HPO was about 5-fold greater than that with cumene hydroperoxide as a substrate. GST A1/A1 was more active with 9 c,t-HPO, whereas GST B1/B2 exhibited equivalent activity with both 9 c,t-HPO and 13 c,t-HPO. 4HNE is a toxic, a,b-unsaturated aldehyde that is generated by peroxidation of arachidonic acid in rat microsomes exposed to oxidative stress (Esterbauer et al., 1986). GST B1/B2 exhibited a low level of GSH conjugation activity with 4HNE (Table III) when measured using the mammalian GST assay of Ålin et al. (1985). However, modifying the assay medium by increasing GSH concentration from 0.5 to 1.0 mm and the pH from 6.5 to 7.5 resulted in about a 2-fold increase in the activity of GST B1/B2 with 4HNE. GST A1/A1 exhibited no activity with this substrate under either set of conditions.

Ethacrynic acid is a phenylacetic acid derivative used as a diuretic (Ahokas et al., 1985). It contains an electrophilic group similar to a-b-alkenals generated in mammals under oxidative stress (Danielson et al., 1987; Berhane et al., 1994). GST A1/A1 and GST B1/B2 exhibited similar activities with this substrate when assayed at a GSH concentration of 0.25 mm (Table III), the concentration typically used to assay the activity of mammalian GSTs with ethacrynic acid (Habig and Jakoby, 1981). However, when the GSH concentration was increased to 1 mm, the activity of GST A1/A1 increased 9-fold, whereas the activity of GST B1/B2 increased only slightly.

Kinetics Bireactant kinetic models were used to evaluate the kinetic mechanism that best described GST A1/A1 and GST B1/B2. For GST A1/A1, we analyzed the initial-velocity data using the random, steady-state Bi-Bi equation (Eq. 2) and the random, rapid-equilibrium Bi-Bi equation (Eq. 1). Initial-velocity data exhibited a poor fit to the random, steady-state model as indicated by large standard errors and negative values for several of the kinetic parameters. The random, rapid-equilibrium model (Eq. 1) provided a better fit of the data (Fig. 5). Multiple intersecting lines were generated in the reciprocal plots for both CDNB and GSH, indicating that the mechanism was sequential. The common intersection point lies below the abscissa (a 5

Table III. Activity of GST A1/A1 and GST B1/B2 with various substrates Values are means 6

SE.

Substrate

[GSH]

pH

GST A1/A1

CDNB (1.00 mM) DCNB (1.00 mM) Metolachlor (0.005 mM) Alachlor (0.005 mM) Atrazine (0.005 mM) EPTC-sulfoxide (0.005 mM) Cumene hydroperoxide (0.100 mM) 9 c,t-Linoleic hydroperoxide (0.100 mM) 13 c,t-Linoleic hydroperoxide (0.100 mM) 4HNE (0.100 mM) Ethacrynic acid (0.200 mM)

1.00 1.00 5.00 1.00 1.00 1.00 1.00 1.00 4.00 4.00 4.00 0.50 1.00 0.25 1.00

GST B1/B2

unitsa mg21

mM

6.5 7.5 7.5 7.5 7.5 7.0 6.5 6.8 7.0 7.0 7.0 6.5 7.5 6.5 6.5

29.27 6 0.92 46.90 6 0.64 0.13 6 0.01 0.29 6 0.06 0.77 6 0.26 7.00 6 0.60 1.11 6 0.24 N.D. 0.34 6 0.11 1.71 6 0.20 0.38 6 0.10 N.D. N.D. 0.26 6 0.04 2.34 6 0.28

7.32 6 0.85 10.41 6 0.53 N.D.b N.D. 54.07 6 1.47 63.37 6 1.86 4.62 6 0.81 7.97 6 0.75 0.28 6 0.12 1.24 6 0.04 1.21 6 0.05 0.07 6 0.01 0.13 6 0.02 0.32 6 0.03 0.41 6 0.03

a 1 unit 5 1 mmol product produced per minute except for the herbicides (metolachlor, alachlor, atrazine, and EPTC-sulfoxide) where 1 unit b 5 1 nmol conjugate produced per hour. N.D., No detectable activity.


Figure 5. Double-reciprocal plots of GST(CDNB) activity for GST A1/A1. The initial-velocity data were fitted to the random, rapidequilibrium Bi-Bi equation (Eq. 1). A, E, 0.02 mM; F, 0.04 mM; M, 0.08 mM; and f, 0.16 mM GSH concentrations; Km for CDNB 5 1.91 mM. B, E, 0.5 mM; F, 1.0 mM; and M, 2.0 mM CDNB concentrations; Km for GSH 5 0.118 mM. v is expressed as micromoles per minute.

1.8), which indicates that binding of the first substrate (GSH or CDNB) decreases the enzyme’s affinity for the second substrate (Segel, 1975). This a value is similar to that of a human placental GST (a 5 2.1), which was also best described by the random, rapid-equilibrium model (Ivanetich and Goold, 1989). For GST A1/A1, the Km values for GSH and CDNB were 118 mm (Ks 5 65 mm) and 1913 mm (Ks 5 1063 mm), respectively. Although the random, rapidequilibrium model provided a good fit to the initialvelocity data for GST A1/A1 (Fig. 5), additional experiments involving end-product inhibition would be required to definitively demonstrate a random, rapid-equilibrium mechanism (Segel, 1975). Reciprocal plots of the initial-velocity data for the heterodimer GST B1/B2 using the substrates GSH and metolachlor were nonlinear (data not shown). Attempts at fitting the initial-velocity data to the random, rapidequilibrium model (Eq. 1) did not yield a random distribution of residuals. The data were then analyzed using the random, steady-state equation (Eq. 2) because the presence of the squared terms in this model predicts

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nonlinear, reciprocal plots (Segel, 1975). Nonlinear, reciprocal initial-velocity plots have been observed for rat liver GST 2–2 and GST 3–3 (Jakobson et al., 1979; Ivanetich et al., 1990), and the initial-velocity data for these isozymes were fitted to the random, steady-state model. However, attempts to fit the initial-velocity data for GST B1/B2 to this model generated parameter values with high standard errors and multiple negative values. The fact that GST B1/B2 is a heterodimer suggested another possible explanation for the biphasic kinetic data. Multisite enzymes with different affinities for the same substrate will yield nonlinear, reciprocal plots (Segel, 1975). Although multisite kinetic analysis was developed to determine kinetic constants for a mixture of two enzymes acting on the same substrate, it is also applicable for one enzyme that exhibits two binding sites differing in substrate affinity (Segel, 1975). It has been established that subunits of mammalian GST heterodimers are catalytically independent and that kinetic constants are additive (Danielson and Mannervik, 1985; Tahir and Mannervik, 1986). Therefore, the possibility was considered that the biphasic pattern for the initial-velocity data for GST B1/B2 reflects the contributions of two subunits that exhibit different affinities for the substrates GSH and metolachlor. Plotting the initial-velocity data for GST B1/B2 on EadieHofstee plots (Fig. 6) yielded a biphasic pattern, suggesting a multisite enzyme with two substrate-binding sites with different affinities (Segel, 1975). Initial estimates of the kinetic parameters for each site were determined by fitting a straight line to the two linear portions of the graph. Kinetic parameters for the high-affinity site were derived from the straight lines generated by linear regression below GSH and metolachlor concentrations of 60 and 20 mm, respectively. For the low-affinity site, kinetic parameters were derived from the straight lines generated by linear regression for GSH and metolachlor concentrations greater than 320 and 80 mm, respectively. This approach yielded estimated Km values of 16 and 5 mm for GSH and metolachlor, respectively, at the high-affinity site, and 370 and 286 mm, respectively, at the low-affinity site. Derivation of these parameters directly from the linear portions of the graph assumes that at low substrate concentrations the low-affinity site does not contribute significantly to the measured velocity and that at high substrate concentrations, the high-affinity site does not contribute significantly to the measured velocity. However, this assumption generally does not hold true (Spears et al., 1971), which was found to be the case for GST B1/B2. Starting with the initial estimates of kinetic parameters for the high-affinity site, three rounds of successive corrections as described by Spears et al. (1971) were performed to calculate the corrected Km values for both sites. For the highaffinity subunit of GST B1/B2, this procedure yielded Km values for GSH and metolachlor of 12 and 3 mm, respectively. The Km values for GSH and metolachlor for the low-affinity subunit of GST B1/B2 were 915 and 421 mm, respectively. The calculated Vmax values for GST(metolachlor) activity of the low- and high-affinity subunits were 0.14 and 2.19 nmol h21, respectively. As indicated in Figure 6, the predicted velocities calculated using the corrected

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Figure 6. Eadie-Hofstee plots for GST(metolachlor) activity for GST B1/B2. The dashed lines were generated by three rounds of successive corrections, as described by Spears et al. (1971), and used to calculate corrected Km values for the low- and high-affinity sites. A, Metolachlor was varied from 5 to 640 mM at a saturating, fixed GSH concentration of 1280 mM. The Km values for metolachlor at the low- and high-affinity sites were 421 and 3 mM, respectively. B, GSH concentration was varied from 20 to 1280 mM at a saturating, fixed metolachlor concentration of 640 mM. The Km values for GSH at the lowand high-affinity sites were 915 and 12 mM, respectively. For both A and B, the solid, curved line is the predicted velocity calculated when the corrected kinetic constants for the low- (short dashes) and high-affinity (long dashes) sites are substituted into Equation 3. v is expressed as nanomoles per hour.

kinetic constants (Eq. 3) are in close agreement with the measured velocities.

I50 Values The effect of inhibitors of mammalian GSTs on the activity of GST A1/A1 and GST B1/B2 was examined (Table IV). GST A1/A1 was assayed with CDNB as the substrate, whereas GST B1/B2 was assayed using metolachlor as the substrate. Under our assay conditions for GST(metolachlor) activity (5 mm metolachlor, 1 mm GSH), we were primarily measuring the inhibitor sensitivity of the highaffinity subunit of GST B1/B2, which contributed approximately 75% of the observed velocity. Of the GST inhibitors examined, Cibracon blue was the most potent and, in general, the heterodimer GST B1/B2 was less sensitive to the inhibitors.

reagent, which detects the presence of unsubstituted vicinyl hydroxyls of Man, Glc, and Gal (Dyer, 1956). Purified GST A1/A1 and GST B1/B2 were electrophoresed on SDSPAGE Phast gels, blotted onto Immobilon P membranes, and treated with the PAS reagent, as described by Stro¨mqvist and Gruffman (1992). Glycosylation was not detected for either isozyme using this method (data not shown). However, glycosylation of GST A1/A1 (Fig. 7, lane 1) and GST B1/B2 (data not shown) was demonstrated using ConA-biotin/avidin-alkaline phosphatase. ConA binds specifically to Man and Glc residues and to glucosamine with lower affinity (Poretz and Goldstein, 1970). ConA-biotin, which contains six molecules of biotin per

Glycosylation Initial tests to determine whether GST A1/A1 and GST B1/B2 were glycosylated were conducted using the PAS Table IV. I50 values for GST A1/A1 and GST B1/B2 measured with selected inhibitors Values are means 6 SE. Inhibitor

GST A1/A1a

S-hexyl-GSH Chlorotriphenyltin Sulfobromophthalein Cibacron blue

1048 6 42 24.9 6 1.4 7.1 6 0.5 0.57 6 0.03

GST B1/B2b

mM

a

6520 6 339 39.7 6 2.7 41.7 6 5.8 5.74 6 0.07

Assayed using CDNB and GSH as the substrates. using metolachlor and GSH as the substrates.

b

Assayed

Figure 7. Western blot of denatured GST A1/A1 (6 a-mannosidase treatment) probed with ConA-biotin/avidin-alkaline phosphatase. Lane 1, GST A1/A1 without a-mannosidase treatment; lane 2, GST A1/A1 pretreated with a-mannosidase. The amount of GST A1/A1 protein per lane was 200 ng. The position of the GST A1 subunit at 26 kD is indicated by the arrowhead on the left. The position of the heavy subunit of a-mannosidase, a mannosylated glycoprotein with a Mr of 68,000, is indicated by the arrowhead on the right.


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acterize GST A1/A1 and GST B1/B2. A single protein peak was observed for the homodimeric GST A1/A1 and two peaks of equal height were observed for the heterodimeric GST B1/B2. The retention times for the 26-kD B1 subunit of GST B1/B2 and the 26-kD subunit of GST A1/A1 were similar. Western blots of the HPLC-purified subunits were probed with ConA-biotin (Fig. 9A), and on the basis of degree of ConA-biotin binding, the GST A1 subunit was shown to be the least glycosylated and the GST B2 subunit the most heavily glycosylated.

Antigenic Cross-Reactivity Further evidence of the heterodimeric nature of GST B1/B2 was the difference in cross-reactivity of the B1 and B2 subunits with antibodies to GST A1/A1. GST A1/A1 antibodies cross-reacted with the 26-kD B1 subunit but not the 28-kD B2 subunit (Fig. 9B). Antibodies to the GST A1 subunit recognized other 26-kD subunits found in the multiple GST peaks in fluxofenim-treated sorghum shoots (data not shown). A difference in antigenic cross-reactivity of GST subunits of heterodimers has been observed for maize I/II (formerly GST II; see Dixon et al., 1997, for new nomenclature for maize GSTs). For maize GST I/II, antibodies generated to one subunit did not cross-react with the other subunit (Holt et al., 1995). Figure 8. Purification of subunits from GST A1/A1 (A) and GST B1/B2 (B) by HPLC reverse-phase chromatography. Native GST A1/A1 or B1/B2 was applied to a reverse-phase HPLC column and eluted with a gradient of water and trifluoroacetic acid in acetonitrile as described in “Materials and Methods.” Background absorbance due to increasing trifluoroacetic acid concentration during the gradient has been subtracted.

molecule of ConA, allows for low levels of glycosylation to be visualized. Other studies have demonstrated the greater sensitivity of ConA for detecting glycoproteins compared with the PAS method (Wood and Sarinana, 1975; Bayer et al., 1987; Kuzmich et al., 1991). Further evidence for glycosylation of GST A1/A1 (Fig. 7, lane 2) and GST B1/B2 (data not shown) was the lack of detection by ConA-biotin/avidin-alkaline phosphatase when the enzyme was pretreated with a-mannosidase. The binding of ConA was specific for glycan moieties because the addition of methylmannoside, a competitive ligand for ConA, decreased by greater than 90% the amount of ConAbiotin that bound to either GST A1/A1 or GST B1/B2 during western analysis (data not shown). Cleavage of terminal Man residues by a-mannosidase did not alter the Mr of the GST A1, B1, and B2 subunits, as indicated by the lack of a mobility shift on SDS-PAGE after treatment (data not shown), suggesting that the extent of mannosylation of these GST subunits was minor. Reverse-phase HPLC chromatography was used to separate GST A1/A1 and GST B1/B2 subunits prior to comparing the degree of glycosylation. Purified native GST A1/A1 or GST B1/B2 was applied to a reverse-phase HPLC column and separated using a gradient of water and trifluoroacetic acid in acetonitrile (Fig. 8). The HPLC profiles verify the purity of the protein fractions used to char-

N-Terminal Sequence Homology N-terminal sequences obtained by Edman degradation for GST A1, B1, and B2 subunits were compared with sequences for type I plant GSTs (Fig. 10), which contain the majority of published sequences for grass GSTs (Droog et al., 1995; Marrs, 1996). The N-terminal sequences for the sorghum GST A1, B1, and B2 subunits are highly homolo-

Figure 9. Western blots of reverse-phase, HPLC-purified subunits of GST A1/A1 and GST B1/B2 probed with ConA-biotin/avidin-alkaline phosphatase (A) and GST A1/A1 antibody/anti-chicken IgG-alkaline phosphatase (B). Lane 1, GST B2; lane 2, GST B1; and lane 3, GST A1. The amount of GST protein per lane was 75 ng.

888


Figure 10. Comparison of N-terminal amino acid sequences of sorghum GST A1, B1, and B2 subunits with other plant type-I GSTs. Alignments were performed using the BESTFIT program of the GCG package (Genetics Computer Group, Madison, WI). A line denotes 100% sequence identity with GST A1, two dots denote an acceptable amino acid substitution, one dot denotes a less acceptable amino acid substitution, and X denotes an unidentified amino acid residue. Acceptable and less acceptable amino acid substitutions are defined by the GCG software. Sequences from sorghum were compared with GSTs from sugarcane (Singhal et al., 1991), maize GST I, (Shah et al., 1986), maize GST III (Grove et al., 1988), maize GST IV (GST II, new nomenclature; Jepson et al., 1994; Dixon et al., 1997), wheat GST A1 (Dudler et al., 1991), Hyoscyamus muticus (Bilang et al., 1993), Arabidopsis pm239 (Bartling et al., 1993), tobacco parB (Takahashi and Nagata, 1992), Arabidopsis pm24 (Zhou and Goldsbrough, 1993), Silene cucubalus (Kutchan and Hochberger, 1992), and Arabidopsis ERD11 (Kiyosue et al., 1993).

gous to each other. The N-terminal sequences of the sorghum GST subunits also exhibited a high degree of homology with maize GST I (GST I/I, new nomenclature) and a GST subunit from sugarcane. A lesser degree of homology was observed for maize GST III (GST III/III, new nomenclature) and GST IV (GST II/II, new nomenclature). Sorghum GSTs exhibited the least homology with the dehydration-induced GST from Arabidopsis (ERD11). DISCUSSION Two GST isozymes, GST A1/A1 (a homodimer) and GST B1/B2 (a heterodimer), were purified to homogeneity from fluxofenim-treated sorghum shoots. The two isozymes were glycosylated as indicated by their binding of ConAbiotin and exhibited GSH peroxidase activity with cumene hydroperoxide and linoleic acid hydroperoxides. Kinetic analysis indicated that GSTA1/A1 was best described by a random, rapid equilibrium Bi-Bi model for the substrates GSH and CDNB. In contrast, the best description of the kinetics of the GST B1/B2 heterodimer for the substrates GSH and metolachlor was provided by a multisite model that allowed for the determination of kinetic constants for each subunit.


To our knowledge the results obtained with GST A1/A1 and B1/B2 are the first reports of glycosylation of plant GSTs. There have been few investigations of posttranslational modification of GSTs. In vitro phosphorylation and methylation of mammalian GSTs have been reported. Cytosolic rat liver GSTs were phosphorylated by a Ca21phospholipid-dependent protein kinase from rabbit brain (Taniguchi and Pyerin, 1989). In vitro, calmodulinstimulated (Johnson et al., 1990) and methyltransferasecatalyzed methylation (Johnson et al., 1992) of rat liver cytosolic GSTs has been reported. There is one report of glycosylated GSTs in mammals. Human GST p and rat GST Yp were detected as glycoproteins by visualization with fluorescein isothiocyanate-ConA, a fluorescent ConA conjugate (Kuzmich et al., 1991). As with sorghum GST A1/A1 and GST B1/B2, the rat liver and human GSTs were not heavily glycosylated because they were not detected by procedures that utilized periodate oxidation for visualization (Kuzmich et al., 1991). Glycosylation of sorghum GST subunits was not limited to GST A1/A1 and GST B1/B2. Each of the eight peaks of GST activity in safener-induced sorghum shoots (Fig. 1B) contained at least one glycosylated subunit, as indicated by visualization with ConAbiotin (data not shown). The function of glycosylation of sorghum GSTs is not known. Previous research has demonstrated that glycosylation of proteins can play a role in proper protein folding, protection against proteases, solubility, and recognition phenomena (Varki, 1993). Further research is needed to determine whether glycosylation of plant GSTs is widespread and, if so, to determine its function. In all previous determinations of kinetic parameters for purified plant GSTs, initial-velocity data have been analyzed using the standard Michaelis-Menton equation for a unireaction (O’Connell et al., 1988; Williamson and Beverley, 1988; Singhal et al., 1991; Irzyk and Fuerst, 1993; Droog et al., 1995; Flury et al., 1995). However, kinetic models describing bireactant mechanisms are better suited for the determination of kinetic constants for GSTs, which utilize two substrates. For GST A1/A1, the random, rapidequilibrium Bi-Bi model best described the kinetics with GSH and CDNB as substrates. Although the results were consistent with this model, further kinetic analysis involving end-product inhibition would be required to confirm the random, rapid equilibrium Bi-Bi model. Bireactant models have been used to characterize the kinetic mechanisms of mammalian GSTs (Jakobson et al., 1979; Schramm et al., 1984; Ivanetich and Goold, 1989; Young and Briedis, 1989; Ivanetich et al., 1990; Phillips and Mantle, 1991). For mammalian GSTs, analyses of initialvelocity data with bireactant kinetic models have indicated that the kinetic mechanism is random and sequential. However, there is a lack of agreement as to whether mammalian GSTs exhibit rapid-equilibrium or steady-state kinetics. In some cases, the random, rapid-equilibrium model provided the best fit to the data (Schramm et al., 1984; Ivanetich and Goold, 1989; Young and Briedis, 1989; Phillips and Mantle, 1991), whereas in others the kinetics were best described by the random, steady-state model (Jakobson et al., 1979; Ivanetich et al., 1990). It has been suggested

Sorghum Glutathione S-Transferase Isozymes that the kinetic mechanism for mammalian GSTs is isozyme specific (Ivanetich and Goold, 1989). For GST B1/B2, bireactant kinetic models did not provide a good fit to the initial-velocity data. A better description of the kinetics was obtained with a multisite enzyme analysis in which kinetic constants for each subunit could be determined by successive correction (Spears et al., 1971). This analysis provided evidence for two catalytically distinct subunits differing in substrate affinities. The results are consistent with reports of catalytic independence of the subunits of mammalian GSTs (Danielson and Mannervik, 1985; Tahir and Mannervik, 1986). In some respects, GST B1/B2 is similar to the maize heterodimer GST I/II (formerly GST II). Both heterodimers contain a herbicide safener-induced subunit that exhibits high affinity for chloroacetanilide herbicides (Irzyk and Fuerst, 1993; Holt et al., 1995; Dixon et al., 1997). The Km values for metolachlor for the high-affinity subunit of GST B1/B2 and the maize GST II subunit are 3 mm (Fig. 8) and 10.8 mm (Irzyk and Fuerst, 1993; GST IV, new nomenclature GST II/II), respectively. These subunits, by exhibiting high affinity for chloroacetanilide herbicides, would allow for detoxification of micromolar concentrations of these herbicides, which are inhibitory to plant growth (Deal and Hess, 1980; Fuerst and Gronwald, 1986; Fuerst et al., 1991). These Km values contrast sharply with the apparent Km for metolachlor of 8.9 mm for the GST III/III (formerly GST III) isozyme found in maize coleoptiles (O’Connell et al., 1988). Although the maize GST II subunit and the high-affinity subunit of sorghum GST B1/B2 both exhibit high affinity for chloroacetanilides, they differ in affinity for GSH. The high-affinity subunit of GST B1/B2 exhibited a Km of 12 mm for GSH, whereas the GST II subunit exhibited an apparent Km of 292 mm (Irzyk and Fuerst, 1993). The Km of the high-affinity subunit of B1/B2 for GSH is one of the lowest values reported in the literature for either plant or mammalian GSTs. For mammalian GSTs an apparent Km for GSH of 27 mm was reported for rat GST 4–4 with CDNB as a substrate (Zhang et al., 1992). A heterologously expressed GST from Arabidopsis exhibited an apparent Km of 80 mm for GSH when assayed with CDNB (Bartling et al., 1993). With the exception of the pathogen-induced GSTA1 from wheat and GST ERD11 from Arabidopsis, type-I u-GSTs have a conserved Ser in the region of residues 10 through 13 near the N terminus (domain I) (Fig. 10). There is increasing evidence that an N-terminal Ser of u-GSTs plays a critical role in catalysis and is equivalent to the domain I, N-terminal Tyr of mammalian a-, m-, and p-GSTs that is involved in the formation of the thiolate anion of bound GSH (Blocki et al., 1993; Dirr et al., 1994). Site-directed mutagenesis studies of the u-GST from the Australian sheep blowfly (Lucilia cuprina) and the u-GST from the bacterium Methylophilus sp. strain DM11 indicated that the Ser-9 and Ser-12 residues, respectively, are essential for enzyme activity (Board et al., 1995; Vuilleumier and Leisinger, 1996). Furthermore, the three-dimensional structure of a u-GST from L. cuprina showed that Ser-9 occupies a position close to that of the catalytically important Tyr of mammalian a-, m-, and p-GSTs (Wilce et al., 1995). Recently, the first three-dimensional structure of a plant GST

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was reported (Reinemer et al., 1996). This enzyme, a type I GST from Arabidopsis (pm24; Fig. 10), contains no Tyr residue in the active site. However, like the GST from L. cuprina, it contains a Ser residue that is in a location similar to that of the catalytically active Tyr in mammalian GSTs. Further research involving site-directed mutagenesis and x-ray crystallography analysis is needed to evaluate the role of the N-terminal Ser residue in the mechanism of catalysis by u-GSTs. Both GST A1/A1 and GST B1/B2 exhibited activity with ethacrynic acid, a phenylacetic acid derivative that contains an electrophilic group similar to a-b-alkenals generated in mammals under oxidative stress (Danielson et al., 1987; Berhane et al., 1994). Ethacrynic acid can act as a substrate (Yamada and Kaplowitz, 1980), activator (Phillips and Mantle, 1991), or inhibitor (Ahokas et al., 1985; Phillips and Mantle, 1993) of mammalian GSTs, depending on the isozyme. High activity with ethacrynic acid is characteristic of mammalian p-GSTs (Mannervik and Danielson, 1988). In pea epicotyls GST activity with ethacrynic acid was inducible by treatment with GSH and a fungal elicitor (Edwards, 1996). In contrast, ethacrynic acid was a potent inhibitor (Ki 5 5 mm) of auxin-inducible GSTs from tobacco (Droog et al., 1995). GST B1/B2, but not GST A1/A1, exhibited GSHconjugating activity with 4HNE, a toxic a-b-unsaturated aldehyde that is generated by peroxidation of arachidonic acid in rat microsomes exposed to oxidative stress (Esterbauer et al., 1986). In rat liver, the a-GST 8–8 exhibits high activity with this substrate (Danielson et al., 1987). There have been no previous reports of plant GSTs exhibiting activity with 4HNE. In plants alkenals such as trans-2hexenal are derived from 13-hydroperoxylinolenic acid after cleavage with hydroperoxide lyase (Gardner, 1991). An Arabidopsis GST active with 13-hydroperoxylinolenic acid exhibited no activity with trans-2-hexenal (Bartling et al., 1993). Both GST A1/A1 and B1/B2 exhibited GSH peroxidase activity with cumene hydroperoxide and linoleic acid hydroperoxides. There have been an increasing number of reports of plant GSTs exhibiting GSH peroxidase activity with these substrates. In pea, GSH peroxidase activity with cumene hydroperoxide was higher in etiolated seedlings than in older green plants and was induced in roots by treatment with CuCl2 (Edwards, 1996). An auxin-binding, plasma membrane-associated GST from Arabidopsis exhibited activity with cumene hydroperoxide (Zettl et al., 1994). GSH peroxidase activity with lipid hydroperoxides as substrates has been reported for GSTs from pea seeds (Williamson and Beverley, 1987), wheat flour (Williamson and Beverley, 1988), soybean hypocotyls (Flury et al., 1996), and Arabidopsis (Bartling et al., 1993). Compared with the GSTs purified from pea seeds and wheat flour, sorghum GST A1/A1 and GST B1/B2 exhibit higher activity with both 9 c,t-HPO and 13 c,t-HPO. Similar to sorghum GST B1/B2, a GST purified from pea seeds exhibited equivalent GSH peroxidase activity with 9 c,tHPO and 13 c,t-HPO (Williamson and Beverley, 1987). In contrast, a GST isolated from wheat flour exhibited a preference for 9 c,t-HPO over 13 c,t-HPO (Williamson and

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Beverley, 1988) similar to what was observed for sorghum GST A1/A1 (Table III). The level of GSH peroxidase activity of GST A1/A1 and GST B1/B2 with linoleic hydroperoxides is similar to that reported for linolenic and arachidonic hydroperoxides (0.4–1.2 mmol min21 mg21) in a purified GST fraction from soybean hypocotyls (Flury et al., 1996). GSH peroxidase activity of GST A1/A1 and B1/B2 with linoleic hydroperoxides is within the range of activities reported for mammalian a-GSTs, which are distinguished by their activity with products of oxidative stress (Ketterer and Coles, 1991). Plant GSTs are members of the archaic u-GST class, from which other GST classes evolved (Buetler and Eaton, 1992). It has been proposed that u-GSTs originally evolved in prokaryotes to protect against oxidative stress (Pemble and Taylor, 1992). In plants, oxidative stress may signal the induction of certain GSTs (Flury et al., 1996; Marrs, 1996). Several treatments known to induce GSTs, such as wounding or exposure to ozone, ethylene, heavy metals, or pathogens, cause oxidative stress (Marrs, 1996, and refs. therein). Safener treatment of sorghum causes stress, as indicated by growth inhibition of developing seedlings (Fuerst and Gronwald, 1986), but why safeners inhibit growth is not known. Although the safener-induced GST B1/B2 exhibits activity with herbicidal concentrations of metolachlor, it is possible that the primary function of this isozyme, and perhaps others induced by the safener, is to protect against lipid peroxidation products generated by various forms of stress. At comparable substrate concentrations (4 mm GSH and 0.1 mm metolachlor or 13 c,t HPO), the activity of GST B1/B2 with 13 c,t-HPO is at least 2 orders of magnitude greater than that with metolachlor as a substrate. It would be of interest to determine whether treatments that cause oxidative stress, such as exposure to ozone and hydrogen peroxide, would induce GST B1/B2 in sorghum, and whether pretreating sorghum with fluxofenim would confer protection against subsequent exposure to oxidative stress. ACKNOWLEDGMENTS We express our appreciation to Novartis for providing sorghum seeds and 14C-labeled herbicides (metolachlor, alachlor, and atrazine). 4HNE was generously provided by Dr. H. Esterbauer (Institut fu¨r Biochemie, Universitat Graz, Graz, Austria). Received November 5, 1997; accepted March 25, 1998. Copyright Clearance Center: 0032–0889/98/117/0877/16. LITERATURE CITED Ahokas JT, Nicholls FA, Ravenscroft PJ, Emmerson BT (1985) Inhibition of purified rat liver glutathione S-transferase isozymes by diuretic drugs. Biochem Pharmacol 34: 2157–2161 Ålin P, Danielson UH, Mannervik B (1985) 4-Hydroxyalk-2-enals are substrates for glutathione transferase. FEBS Lett 179: 267–270 Awasthi YC, Beutler E, Srivastava SK (1975) Purification and properties of human erythrocyte glutathione peroxidase. J Biol Chem 250: 5144–5149 Bartling D, Radzio R, Steiner U, Weiler EW (1993) A glutathione S-transferase with glutathione-peroxidase activity from Arabidopsis thaliana. Molecular cloning and functional characterization. Eur J Biochem 216: 579–586


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