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

	ABSTRACT

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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, rapid-equilibrium 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 -GSTs indicated a high degree of homology with the maize GST I subunit and a sugarcane GST.

	INTRODUCTION

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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 ligand-binding 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 (, µ, , and ) based on immunological, 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 -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 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 -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 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

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Chemicals²

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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 für 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 × 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 incremental 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

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GST Dimer and Monomer Purification

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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-hexyl-GSH in 0.2 M NaCl at a flow rate of 1 mL min¹. 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 M_r 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 min¹. 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.

View larger version (32K): [in this window] [in a new window]   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 () at pH 7.5 and for GST(metolachlor) activity () as described in ``Materials and Methods''.

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 min¹ was used to resolve GST proteins. Protein was detected at 220 nm.

Enzyme Assays

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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 µM [¹⁴C]herbicide (specific activity for metolachlor and alachlor, 5 µCi µmol¹; for EPTC-sulfoxide, 2 µCi µmol¹; and for atrazine, 4.5 µCi µmol¹), 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-µL 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 EPTC-sulfoxide, 1 unit of activity is defined as the formation of 1 nmol conjugate h¹ 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 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 µg) 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

(1)

(2)

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 µM, respectively, were used to generate constants for the high-affinity site, and concentrations greater than 320 and 80 µM, respectively, were used for determining kinetic constants for the low-affinity site. Linear regression was used to fit the data in the defined concentration ranges to a straight line and kinetic constants (K_m and V_max) 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 µM were used for the high-affinity site and those above 100 µM for the low-affinity site. For metolachlor, substrate concentrations of less than 50 µM were used for the high-affinity site and those greater than 50 µM were used for the low-affinity site. The initial estimates of K_m and V_max 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 V_max and K_m 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 V_max and K_m for the high-affinity 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:

(3)

where V₁, V_max1, and K_m1 are kinetic parameters for the corrected low-affinity site, and V₂, V_max2, and K_m2 are the corrected kinetic parameters for the high-affinity site.

Native Molecular Mass

Gel Electrophoresis

Determination of I₅₀ Values

-Mannosidase Treatment

Detection of Glycosylation

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Polyclonal Antibody Production

Antibody Cross-Reactivity

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N-Terminal Sequencing

Protein Determination

	RESULTS

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Purification of GST A1/A1 and B1/B2

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 trend toward less induction of GST(CDNB) activity of peak 2 relative to peak 1 was observed with aging of fluxofenim-treated 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 µM). 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 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.

View larger version (31K): [in this window] [in a new window]   Figure 2. Chromatofocusing of peak 2 (Fig. 1) from untreated (A) and fluxofenim-treated (B) sorghum shoots. Fractions were assayed for GST(CDNB) (), GST(metolachlor) (), and GST(alachlor) () 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.

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 SDS-PAGE, 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).

View larger version (50K): [in this window] [in a new window]   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.

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 M_r 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 M_r for GST B1/B2 is close to the calculated value of 54,000. However, the native M_r 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.

View larger version (40K): [in this window] [in a new window]   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.

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 safener-treated, 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

GST A1/A1 exhibited relatively low activity with the chloroacetanilide herbicides metolachlor and alachlor but 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, ,-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 --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

View larger version (22K): [in this window] [in a new window]   Figure 5. Double-reciprocal plots of GST(CDNB) activity for GST A1/A1. The initial-velocity data were fitted to the random, rapid-equilibrium Bi-Bi equation (Eq. 1). A, , 0.02 mM; , 0.04 mM; , 0.08 mM; and , 0.16 mM GSH concentrations; Km for CDNB = 1.91 mM. B, , 0.5 mM; , 1.0 mM; and , 2.0 mM CDNB concentrations; Km for GSH = 0.118 mM. v is expressed as micromoles per minute.

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, rapid-equilibrium 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 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 Eadie-Hofstee 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 µM, 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 µM, respectively. This approach yielded estimated K_m values of 16 and 5 µM for GSH and metolachlor, respectively, at the high-affinity site, and 370 and 286 µM, respectively, at the low-affinity site.

View larger version (15K): [in this window] [in a new window]   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 µM at a saturating, fixed GSH concentration of 1280 µM. The Km values for metolachlor at the low- and high-affinity sites were 421 and 3 µM, respectively. B, GSH concentration was varied from 20 to 1280 µM at a saturating, fixed metolachlor concentration of 640 µM. The Km values for GSH at the low- and high-affinity sites were 915 and 12 µM, 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.

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 K_m values for both sites. For the high-affinity subunit of GST B1/B2, this procedure yielded K_m values for GSH and metolachlor of 12 and 3 µM, respectively. The K_m values for GSH and metolachlor for the low-affinity subunit of GST B1/B2 were 915 and 421 µM, respectively. The calculated V_max values for GST(metolachlor) activity of the low- and high-affinity subunits were 0.14 and 2.19 nmol h¹, respectively. As indicated in Figure 6, the predicted velocities calculated using the corrected kinetic constants (Eq. 3) are in close agreement with the measured velocities.

I₅₀ Values

Glycosylation

View larger version (52K): [in this window] [in a new window]   Figure 7. Western blot of denatured GST A1/A1 (± -mannosidase treatment) probed with ConA-biotin/avidin-alkaline phosphatase. Lane 1, GST A1/A1 without -mannosidase treatment; lane 2, GST A1/A1 pretreated with -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 -mannosidase, a mannosylated glycoprotein with a Mr of 68,000, is indicated by the arrowhead on the right.

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 -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 ConA-biotin that bound to either GST A1/A1 or GST B1/B2 during western analysis (data not shown). Cleavage of terminal Man residues by -mannosidase did not alter the M_r 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 characterize 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.

View larger version (14K): [in this window] [in a new window]   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.

View larger version (47K): [in this window] [in a new window]   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.

Antigenic Cross-Reactivity

N-Terminal Sequence Homology

View larger version (48K): [in this window] [in a new window]   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).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

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 ConA-biotin 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 Ca²⁺-phospholipid-dependent protein kinase from rabbit brain (Taniguchi and Pyerin, 1989). In vitro, calmodulin-stimulated (Johnson et al., 1990) and methyltransferase-catalyzed methylation (Johnson et al., 1992) of rat liver cytosolic GSTs has been reported. There is one report of glycosylated GSTs in mammals. Human GST  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 ConA-biotin (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, rapid-equilibrium 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 initial-velocity 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 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 K_m values for metolachlor for the high-affinity subunit of GST B1/B2 and the maize GST II subunit are 3 µM (Fig. 8) and 10.8 µM (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 K_m values contrast sharply with the apparent K_m 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 K_m of 12 µM for GSH, whereas the GST II subunit exhibited an apparent K_m of 292 µM (Irzyk and Fuerst, 1993). The K_m 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 K_m for GSH of 27 µM 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 K_m of 80 µM 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 -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 -GSTs plays a critical role in catalysis and is equivalent to the domain I, N-terminal Tyr of mammalian -, µ-, and -GSTs that is involved in the formation of the thiolate anion of bound GSH (Blocki et al., 1993