Enhanced Catalytic Constant for GlutathioneS-Transferase (Atrazine) Activity in an Atrazine-ResistantAbutilon theophrastiBiotype

Pesticide Biochemistry and Physiology 63, 34–49 (1999) Article ID pest.1998.2387, available online at http://www.idealibrary.com on

Enhanced Catalytic Constant for Glutathione S-Transferase (Atrazine) Activity in an Atrazine-Resistant Abutilon theophrasti Biotype Kathryn L. Plaisance* and John W. Gronwald†,1 *Department of Agronomy and Plant Genetics, University of Minnesota, and †Plant Science Research Unit, U. S. Department of Agriculture, Agricultural Research Service, St. Paul, Minnesota 55108 Received May 28, 1998; accepted October 20, 1998 Glutathione S-transferases (GST, EC 2.5.1.18) were purified from leaves of atrazine-resistant and -susceptible velvetleaf (Abutilon theophrasti Medic.) biotypes using a protocol involving DEAE anionexchange, S-hexylglutathione affinity, and Superose 12 gel-filtration chromatography. This protocol resulted in greater than 500-fold purification of GST activity with atrazine [GST (atrazine) activity] from both biotypes. There were no differences in the amount of dimeric GST (55,000 Mr) from resistant and susceptible biotypes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified GST dimers from both biotypes indicated the presence of three GST subunits with Mr of 27,000, 26,000, and 25,000. GST subunits in both resistant and susceptible biotypes were glycosylated as revealed by probing Western blots with concanavalin A-biotin/avidin-alkaline phosphatase. Native isoelectric focusing of the purified GSTs indicated three major isoforms in both biotypes with pI values of 4.1, 4.3, and 4.4. For both resistant and susceptible biotypes, analysis of initial-velocity GST(atrazine) activity of the purified GST fraction indicated a sequential, random, rapid-equilibrium, Bi Bi kinetic mechanism. Kinetic analysis of GST(atrazine) activity from resistant and susceptible biotypes indicated no significant difference in Km values for GSH and atrazine. However, the catalytic constant (kcat) was approximately threefold greater for GST(atrazine) activity from the resistant biotype compared to the susceptible biotype. Inhibition constants (Ki values) for the glutathione-atrazine conjugate were approximately 1.5-fold higher for GST(atrazine) activity from the resistant biotype compared to the susceptible biotype. Accelerated atrazine detoxification via glutathione conjugation in the resistant biotype is primarily due to enhanced kcat for GST(atrazine) activity. 䉷1999 Academic Press

INTRODUCTION

atrazine detoxification was responsible for atrazine resistance in the Maryland biotype (3,4). Recently, atrazine resistance due to enhanced detoxification via glutathione conjugation was reported for a Wisconsin velvetleaf biotype (5). Cytosolic GSTs (EC 2.5.1.18) are multifunctional, dimeric proteins that catalyze nucleophilic attack of the thiolate anion of GSH at electrophilic centers of various hydrophobic compounds (6). Plant GSTs belong to the diverse theta GST class which also includes mammalian, insect, and bacterial GSTs (7,8). Plants, like

In 1984, an atrazine-resistant biotype of velvetleaf (Abutilon theophrasti Medic.) was discovered in a Maryland field that had been treated with triazine herbicides for several years (1). Typically, atrazine resistance in weeds is caused by a mutation of the plastidic psbA gene that encodes the herbicide target site, the D1 protein of photosystem II (2). However, the 10-fold increase in atrazine resistance of the Maryland biotype was due to the mutation of a single, partially dominant, nuclear gene (1,3). Enhanced GST(atrazine)2 activity resulting in accelerated

glutathione conjugate of atrazine; GS-CDNB, glutathione conjugate of CDNB; GST, glutathione S-transferase; GST (atrazine), glutathione S-transferase activity with atrazine as substrate; GST(CDNB), glutathione S-transferase activity with CDNB as substrate; kcat, catalytic constant (Vmax/total enzyme concentration); Ki, inhibition constant; Mr, relative molecular weight; NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa1,3-diazole; PAS, periodic acid/Schiff; PVPP, polyvinylpolypyrrolidone; S-hexylGSH, S-hexylglutathione.

1

To whom correspondence should be addressed at USDAARS, 411 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108. Fax: (651) 649-5058. E-mail: gronw001@ maroon.tc.umn.edu. 2 Abbreviations used: CAPS, 3-cyclohexylamino-1-propanesulfonic acid; CDNB, 1,chloro-2,4-dinitrobenzene; conA, concanavalin A; EPTC, S-ethyldipropylcarbamothioate; FPLC, fast protein liquid chromatography; GS-atrazine, 34 50048-3575/99 $30.00 Copyright 䉷 1999 by Academic Press All rights of reproduction in any form reserved.

Abutilon GLUTATHIONE S-TRANSFERASES

mammals, contain multiple GST isozymes representing homomeric and heteromeric combinations of GST subunits (7,9–11). The bestcharacterized function of plant GSTs is their role in the detoxification of certain herbicide classes such as the s-triazines (e.g., atrazine), thiocarbamates (e.g., EPTC), choroacetanilides (e.g., metolachlor), and diphenylethers (e.g., fluorodifen) (12). In this capacity, GSTs play an important role in determining herbicide selectivity. More recent investigations have indicated that plant GSTs may play a role in plant response to pathogen invasion (13,14) and oxidative stress (15– 18). In maize, the bronze-2 gene encodes a GST that conjugates anthocyanin prior to transport into the vacuole via a GSH-conjugate carrier protein (19). In an earlier paper (4), we established that atrazine resistance in a velvetleaf biotype found in Maryland was due to enhanced GST(atrazine) activity as measured in crude extracts from leaves and stems. The objectives of this investigation were to develop a protocol to purify GST protein from leaves of atrazine-resistant and -susceptible velvetleaf, and to characterize GST(atrazine) activity in this fraction. The results indicate that atrazine-resistance is due to the presence of GST isoforms that exhibit enhanced kcat with atrazine as substrate. MATERIALS AND METHODS

Chemicals [U- C] atrazine (14.6 ␮Ci mg , 98.6% pure) and technical grade atrazine (97% pure) were provide by Novartis (Greensboro, NC). Peter’s Hydrosol was obtained from W.R. Grace & Co. (Fogelsville, PA). DEAE-Sepharose fast-flow and epoxy-activated Sepharose 6B were obtained from Pharmacia Biotech (Piscataway, NJ). S-hexylGSH was synthesized and linked to Sepharose 6B as described by Mannervik and Guthenberg (20). All other chemicals used were purchased from Sigma Chemical Co. (St. Louis, MO). 14

⫺1

Synthesis of GS-Atrazine Conjugate The GS-atrazine conjugate was prepared using a modification of the protocol of Crayford

35

and Hutson (21). Technical grade atrazine (100 mg) was dissolved in 5 mL of acetone and 0.43 mL of 33% trimethylamine in ethanol was added to the solution. After incubation at room temperature (23⬚C) for 24 h, the acetone was evaporated and the resulting crystals were dissolved in 3 mL of distilled water. A 3-mL aqueous solution containing 283 mg GSH and 54 mg Na2CO3 was added, and the reaction mixture was incubated for 6 h in a shaking water bath at 50⬚C. The reaction mixture was partitioned by shaking for 2 min with 9 mL of CH2Cl2 followed by centrifugation (110g, 5 min). Components of the aqueous and organic layers were separated by reverse-phase TLC on Linear K plates containing fluorescent indicator (Whatman, Clifton, NJ) using methanol:water (20:80). Components were visualized under UV light and with ninhydrin spray. The aqueous layer contained the GSatrazine conjugate as indicated by the presence of a conjugate ring and a positive response with ninhydrin. The aqueous layer was dried down under vacuum and the precipitate was dissolved in 1 mL of methanol:water (20:80). The GSatrazine conjugate was further purified with a 50-cm Partisil 10 ODS-3 reverse-phase HPLC column (Whatman) by using isocratic elution (methanol:water, 20:80) at a flow rate of 3 mL min⫺1. Plant Material The source of seed for the atrazine-resistant and -suceptible velvetleaf (A. theophrasti Medic.) biotypes was described earlier (3). Velvetleaf plants were grown hydroponically as previously described (4). GST Purification Leaves from hydroponically grown plants were harvested at the eight-leaf stage and quickly frozen in liquid nitrogen. Leaf tissue (44 g) was ground to a fine powder in a mortar that had been prechilled with liquid nitrogen. The tissue was then added to 300 mL of extraction buffer that consisted of 100 mM Tricine (pH 8.2), 20 mM 2-mercaptoethanol, 10.4 g PVPP, 2 mM EDTA, 10 mM GSH, 1.25 ␮g mL⫺1 pepstatin A, 6 ␮g mL⫺1 antipain, 1 mM PMSF, 10

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mM potassium metabisulfite, and 10.0 g Amberlite XAD-4. All subsequent operations were conducted at 4⬚C. After homogenization, the extract was centrifuged (100,000g, 1 h) and 100 mL of Buffer A [50 mM Tricine (pH 8.2), 1 mM DTT] was added to the supernatant. The supernatant was applied to an Amberlite XAD4 column (2.6 ⫻ 10.0 cm) equilibrated with Buffer A. The eluent from the XAD-4 column was applied to a DEAE-Sepharose FF column (2.5 ⫻ 45 cm) equilibrated in Buffer A. After the column was washed with 100 mL of Buffer A, GST activity was eluted using a linear NaCl gradient (1 L, 0–400 mM). Fractions (10 mL) were collected at a flow rate of 1.5 mL min⫺1. Fractions containing GST(atrazine) activity were collected and applied to a S-hexylGSH affinity column (2.6 ⫻ 18 cm) equilibrated in Buffer A plus 0.1 M NaCl. The column was washed extensively and GST protein was eluted using Buffer A containing 0.1 M NaCl, 5 mM S-hexylGSH and 10 mM GSH. The fraction containing GST protein was collected and concentrated to less than 0.5 mL using a Centricell-20 (10,000 Mr cutoff, Polyscience, Warrington, PA) and desalted using a FPLC Superose 12 HR 10/ 30 column (Pharmacia Biotech) equilibrated in Buffer A. Glutathione S-Transferase Assays GST(atrazine) and GST(CDNB) activities were measured as described by Anderson and Gronwald (4) with modifications. GST(atrazine) activity was assayed at 25⬚C in a medium containing 100 mM potassium phosphate buffer (pH 6.8), 1 mM GSH, 20 ␮M [14C]atrazine (4.5 ␮Ci ␮mol⫺1), 2% ethanol, and protein in a total volume of 0.5 mL. The reaction was stopped after 1 h by addition of TCA (5% final concentration) and partitioned against methylene chloride. [14C] GS-atrazine in the aqueous phase was measured using liquid scintillation spectrometry. GST(CDNB) was assayed at 25⬚C in a medium containing 100 mM potassium phosphate buffer (pH 6.8), 1 mM GSH, 1 mM CDNB, 1% ethanol, and protein in a total volume of 1.0 mL. Nonenzymatic background rates were subtracted for

all assays. One unit of GST(atrazine) activity is defined as 1 nmol GS-atrazine formed h⫺1 and 1 unit of GST(CDNB) activity is defined as 1 ␮mol GS-CDNB formed min⫺1. Kinetic Analysis For initial-velocity studies, GST(atrazine) activity was measured using the standard assay conditions described above. GSH concentration was varied from 0. 5 to 4.0 mM at fixed concentrations of atrazine which varied from 0.08 to 0.64 mM. The specific activity of atrazine remained constant at 80 ␮Ci ␮mol⫺1 over all concentrations. For each experiment, the amount of GST protein per reaction was constant. However, the amount of protein varied from 1.7 to 3.4 ␮g reaction⫺1 between experiments. The kinetic data presented are representative of the results of replicated experiments. Initial-velocity data were fitted to an equation describing a random, sequential, rapid-equilibrium, Bi Bi mechanism (Eq. [1]).

␯⫽ Vmax [A][B] ␣KAKB ⫹ ␣KA[B] ⫹ ␣KB[A] ⫹ [A][B]

[1]

where ␯ ⫽ initial-velocity, [A] ⫽ concentration of one substrate and [B] ⫽ concentration of the other substrate, KA and KB ⫽ dissociation constants with the free enzyme for substrates A and B, respectively; ␣ ⫽ parameter describing the influence of the binding of one substrate on the binding of the second. Nomenclature and definitions of parameters are those of Segel (22). Kinetic constants were determined by nonlinear regression analysis using the Grafit 3.0 computer program (Erithicus Software Ltd., Staines, UK). For the product inhibition studies, GSH concentration was varied from 0.5 to 4.0 mM at a fixed, nonsaturating atrazine concentration of 0.2 mM, while the atrazine concentration was varied from 0.08 to 0.64 mM at a fixed, nonsaturating GSH concentration of 1.0 mM. The GS-atrazine conjugate concentration was varied from 0 to 0.4 mM in experiments using GST protein from the

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resistant biotype, and from 0 to 0.2 mM in experiments using GST protein from the susceptible biotype. The amount of GST protein per reaction and atrazine specific activity were the same as described above. The initial-velocity data for the product-inhibition studies were fitted to an equation for competitive inhibition (Eq. [2]), and kinetic constants were determined by nonlinear regression as described above.

␯⫽

Vmax[S] Km(1 ⫹ [I ]/Ki) ⫹ [S]

[2]

where Ki ⫽ inhibition constant, [S] ⫽ substrate concentration, and [I ] ⫽ inhibitor concentration. Gel Electrophoresis GST subunits were separated by SDS-PAGE on 8 to 25% gradient Phast gels (Pharmacia Biotech). Native isoforms were separated on Pharmacia Phast gels (IEF 4–6.5). Protocols for separation and silver staining were as described by manufacturer. Glycoprotein Detection GST subunits and native forms were separated by SDS-PAGE Phast gels and IEF Phast gels, respectively, as described above, and then transferred to Immobilon P using 10 mM CAPS (pH 11.0) and 50 mM NaCl as the transfer buffer (23). Blots were blocked overnight with Buffer B, which contained 500 mM NaCl, 80 mM TrisHCl (pH 7.6), and 0.1% Tween 20, and then incubated for 1 h in Buffer B containing 5 ␮g mL⫺1 conA-biotin. After two, 10-min washes in Buffer C (0.05% Tween 20, 137 mM NaCl, 3 mM KCl, 25 mM Tris-HCl, pH 7.4), blots were incubated for 1 h with a 1:5000 dilution of avidin-alkaline phosphatase in Buffer B. Blots were again washed with Buffer C and alkaline phosphatase activity was detected by the addition of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium as described by Blake et al. (24).

Protein Determination Protein was estimated by the method of Bradford (25) using the Bio-Rad protein assay reagent and BSA as a standard. RESULTS

Purification A protocol involving DEAE-Sepharose anion-exchange and S-hexylGSH affinity chromatography followed by gel filtration (Superose 12) chromatography resulted in a 613-fold purification of GST(atrazine) activity from leaves of atrazine-resistant velvetleaf (Table 1). GST protein in the leaves of velvetleaf was approximately 0.05% of total soluble protein. This is considerably less than that found in etiolated maize and sorghum shoots where GSTs constitute 1 to 2% of soluble protein (26,27). The yield was approximately 30% and the GST fraction exhibited a specific activity of approximately 800 nmol h⫺1 mg protein⫺1. The most effective step in the purification protocol was the S-hexylGSH affinity chromatography which resulted in a 400-fold increase in specific activity. However, this step resulted in a 64% loss of activity. The sacrifice of yield for purity has been previously noted when S-hexylGSH affinity chromatography is used to purify plant GSTs (15,28).

FIG. 1. SDS-PAGE of the purification of GST protein from leaves of the atrazine-resistant biotype. Proteins were separated on a 8 to 25% gradient Phast gel and visualized by silver staining. Lane 1 and 5, Mr standards; Lane 2, crude extract; Lane 3, DEAE chromatography; and Lane 4, ShexylGSH affinity/Superose 12 chromatography. Equivalent protein (150 ng) was applied per lane.

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PLAISANCE AND GRONWALD TABLE 1 Purification of GST(Atrazine) Activity from Leaves of Atrazine-Resistant Velvetleaf Fraction

Volume

Crude extract DEAE-Sepharose S-hexylGSH affinity/Superose 12

Total protein

(ml) 400 235 6.5

(mg) 300 186 0.158

Total activity (units) 409.7 374.1 135.6

a

Specific activity

Yield

Purification

(units/mg) 1.4 2.0 858.2

(%) 100 91 33

(fold) 1 1.4 613

Note. Results are representative of five isolations. a nmol GS-atrazine produced h⫺1.

For both resistant (Fig. 1) and susceptible biotypes (data not shown), the purified GST fraction contained three GST subunits of 25,000, 26,000, and 27,000 Mr. The relative proportions of the GST subunits were the same in resistant and susceptible biotypes with the 27,000 and 25,000 Mr bands being the darkest. Gel-filtration (Superose 12) chromatography of the purified GST fraction from both resistant and susceptible biotypes indicated a native molecular weight of approximately 55,000 Mr confirming that the GSTs were dimers. A comparison of GST(atrazine) and GST(CDNB) activity of purified GSTs from atrazine-resistant and -susceptible biotypes is shown in Table 2. The amount of GST dimeric protein was essentially the same in the two biotypes. However, both total and specific GST(atrazine) activities were about 3.5-fold greater in the resistant biotype compared to the susceptible biotype. The yield of GST(atrazine) activity was less from the susceptible biotype.

An attempt was made to purify individual GST isoforms in the S-hexylGSH affinity/ Superose 12 fraction by anion-exchange (MonoQ) chromatography. This column had been used earlier to resolve two major peaks of GST(atrazine) activity in a relatively crude fraction obtained from velvetleaf leaves (4). However, individual isozymes exhibiting GST(atrazine) activity could not be resolved on the Mono-Q column. As in the earlier work with crude fractions (4), two major peaks of GST(atrazine) activity were obtained. The results of SDSPAGE chromatography indicated that each peak contained more than one isozyme. Because individual isozymes could not be resolved and the protocol resulted in a considerable loss of GST(atrazine) activity, further purification using the Mono-Q column was not pursued. During DEAE chromatography of leaf extracts from the susceptible biotype, one peak of GST(atrazine) activity eluted in fractions 50–

TABLE 2 Comparison of GST(Atrazine) and GST(CDNB) Activity of Purified GST Protein from Atrazine-Resistant and -Susceptible Velvetleaf Total activity Biotype

Total GST protein

Resistant Susceptible R/Sb

(mg) 0.158 0.151 1.05

GST (atrazine)

GST (CDNB)

(units)a 135.6 9.1 36.8 7.3 3.68 1.25

Specific activity GST (atrazine)

GST (CDNB)

(units/mg) 858.2 57.6 243.7 48.3 3.52 1.19

Yield GST (atrazine)

Purification

GST (CDNB)

(%) 33 19 1.74

29 22 1.32

GST (atrazine)

GST (CDNB)

(fold) 613 577 519 593 1.18 0.97

Note. GST protein was purified from equivalent weight (44 g) of leaves from resistant or susceptible biotypes. a Units for GST (atrazine) activity were nmol GS-atrazine produced h⫺1; units for GST (CDNB) activity were ␮mol GS-CDNB produced min⫺1. b Ratio of parameter of resistant biotype to that of susceptible biotype.

Abutilon GLUTATHIONE S-TRANSFERASES

62 (approximately 260 mM NaCl) (Peak I, Fig. 2A). In contrast, DEAE chromatography of the extract from the resistant biotype yielded two peaks of GST(atrazine) activity; one (Peak I), which eluted at approximately 260 mM NaCl, and the other (Peak II), which eluted in fractions 41–44 (approximately 200 mM NaCl) (Fig. 2B). The proteins in Peaks I from both resistant and susceptible biotypes (Fig. 2) were subjected to S-hexylGSH affinity chromatography followed by SDS-PAGE. Peak I from both biotypes contained polypeptides of 25, 26, and 27 kDa as expected (Fig. 3 lanes 2 and 4). Gel filtration of the affinity-purified proteins in the Peak I fraction from both biotypes indicated the presence of GST dimers (55,000 Mr). GST activity in Peak I of both resistant and susceptible biotypes was stable and this fraction was purified

39

(Fig. 1, Tables 1 and 2) and further characterized (Figs. 4–9, Table 3). GST (atrazine) activity in Peak II of the resistant biotype (Fig. 2B) was labile and rapidly lost activity during S-hexylGSH affinity chromatography. The predominant band in the Peak II fraction from the resistant biotype contained a 25-kDa polypeptide (Fig. 3, lane 5). Although GST(atrazine) activity was not detectable in fractions 41–44 for the susceptible biotype, S-hexylGSH affinity purification of the proteins in this fraction followed by SDS-PAGE indicated the presence of polypeptides in the range of 25 to 27 kDa with the 25-kDa band being predominant (Fig. 3, lane 3). The affinity-purified GST subunits in fractions 41–44 from both resistant and susceptible biotypes were applied to a gel-filtration column (Superose 12). The GST protein that eluted

FIG. 2. DEAE chromatography of protein from leaf extracts of the atrazine-susceptible (A) and -resistant (B) biotypes. Crude, soluble extract from leaves was passed through an Amberlite XAD-4 column prior to being applied to the DEAE column. Fractions were assayed for GST(atrazine) activity. See Materials and Methods for details.

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SDS-PAGE

FIG. 3. SDS-PAGE of Peaks I and II obtained by DEAE chromatography of crude extracts from leaves of atrazineresistant and -susceptible biotypes (Fig. 2). GSTs in the Peak I fraction, which eluted in fractions 50–62 (approximately 260 mM NaCl), and the Peak II fraction, which eluted in fractions 41–44 (approximately 200 mM NaCl), were purified by S-hexylGSH-affinity chromatography prior to SDSPAGE. Proteins were separated on a 8 to 25% gradient Phast gel and visualized by silver staining. Lanes 1 and 6, Mr standards; Lane 2, Peak I (susceptible biotype); Lane 3, fractions 41–44 (susceptible biotype); Lane 4, Peak I (resistant biotype); Lane 5, Peak II (resistant biotype). Equivalent protein (75 ng) was applied per lane.

exhibited a Mr of approximately 25,000, indicating that these fractions contained GST monomers. Protein determinations indicated that the amount of GST monomeric protein in fractions 41–44 (Fig. 2) from susceptible and resistant biotypes was 24 and 37 ␮g, respectively. The GST monomers represented 16 and 23% of the GST protein in the dimer fractions for the susceptible and resistant biotypes, respectively. The putative GST monomers from both resistant and susceptible biotypes were not generated during DEAE chromatography because they were also detected by gel-filtration (Superose 12) chromatography of crude extracts from leaves (data not shown). Furthermore, the putative monomeric GST does not appear to be due to a monomerdimer equilibrium. Reappyling a purified GST dimer fraction back onto the gel-filtration column did not result in the formation of monomeric GST (data not shown). Because of its labile nature, the putative GST monomer was not further characterized.

Glycosylation of GST subunits in the dimeric fraction was evaluated by probing Westerns with conA-biotin which primarily detects mannose and glucose residues (29). As indicated by silver staining of SDS-PAGE gels, both resistant and susceptible biotypes contained two major subunits of 25 and 27 kDa and a minor subunit of 26 kDa in approximately the same amounts (Fig. 4A). Of the two major subunits, the 27-kDa subunit is more heavily glycosylated than the 25-kDa subunit (Fig. 4B). It is not certain whether the 26-kDa subunit is glycosylated because the conA-biotin probe was not able to resolve it from the 27-kDa subunit. Isoelectric Focusing The purified, dimeric GST fraction was subjected to native IEF gel electrophoresis followed by silver staining (Fig. 5A) and probing with conA-biotin/avidin-alkaline phosphatase (Fig. 5B). There were three major and two minor isoforms in both resistant and susceptible biotypes. The three major isoforms exhibited pIs of 4.1, 4.3, and 4.4 while the two minor isoforms exhibited pIs of 4.8 and 4.9 (Fig. 5A). As indicated by silver staining, there appear to be no differences between resistant and susceptible biotypes in the

FIG. 4. SDS-PAGE of purified GST protein from leaves of atrazine-resistant (R) and -susceptible (S) biotypes. (A) silver-stained gel; (B) Western blot of (A) probed with conAbiotin/avidin-alkaline phosphatase. Equivalent GST protein (90 ng) was applied to each lane.

Abutilon GLUTATHIONE S-TRANSFERASES

FIG. 5. Native IEF gel electrophoresis of purified GST protein from leaves of atrazine-resistant and -susceptible biotypes. (A) silver-stained gel: Lane 1, pI standards; Lane 2, susceptible biotype; Lane 3, resistant biotype. (B) Western blot of (A) probed with conA-biotin/avidin-alkaline phosphatase: Lane 1, susceptible biotype; Lane 2, resistant biotype. Equivalent GST protein (850 ng) was applied to each lane.

number of GST isoforms and the relative amount of each. Probing with conA-biotin revealed that most isoforms are glycosylated. It is uncertain whether only one or both of the isoforms with pIs of 4.4 and 4.3 are glycosylated because of the poor resolution obtained with the conA-biotin probe. Of the two minor isoforms, that with a pI of 4.9 is more heavily glycosylated than the isoform with a pI of 4.8. Kinetics Initial-velocity kinetic analysis of GST(atrazine) activity was performed with the purified GST dimer fraction. The initial-velocity data were fitted to a random, rapid-equilibrium model (Eq. [1]). The double-reciprocal plots generated for both GSH and atrazine for GSTs isolated from either susceptible (Fig. 6) or resistant (Fig. 7) biotypes contained a series of intersecting lines in a mixed-type, slope/intercept pattern. Replots of the ordinate intercept versus the reciprocal of the fixed substrate concentration are linear (Insets, Figs. 6 and 7). The data denote a sequential mechanism where both substrates (GSH and atrazine) must bind before products

41

are released. The pattern of lines in the doublereciprocal plots of Figs. 6 and 7 are those predicted by a random, rapid-equilibrium Bi Bi mechanism. Other kinetic mechanisms were evaluated and found to be inconsistent with the data. A rapid-equilibrium, ordered mechanism was ruled out since the initial-velocity analysis did not indicate a competitive inhibition pattern for one of the substrates (22). A steady-state, random mechanism was eliminated because this model contains squared terms that would have yielded a biphasic double-reciprocal plot as well as hyperbolic curves for the ordinate intercept replots (22). As shown in Figs. 6 and 7, the double-reciprocal plots and replots were linear for both substrates. For some enzymes, isotope exchange can be used to determine whether a random system is rapid-equilibrium or steadystate (22). However, isotope-exchange studies are unsuitable for GSTs because the reaction catalyzed is essentially irreversible. A steadystate, ordered Bi Bi mechanism cannot be distinguished from a rapid-equilibrium, random, Bi Bi mechanism by computer modeling because the rate equations are identical and yield identical double-reciprocal plots (22). However, these two mechanisms can be distinguished by product inhibition analysis. For GST(atrazine) activity from the resistant biotype, no inhibition of activity was observed for GS-atrazine concentrations as high as 0.16 mM when saturating concentrations of atrazine and GSH, 0.64 and 4.0 mM, respectively, were present (data not shown). However, 0.16 mM GS-atrazine caused a 60% inhibition of GST(atrazine) activity from the resistant biotype when GSH and atrazine were at subsaturating concentrations. The inability of GS-atrazine to inhibit the forward reaction at saturating concentrations of both substrates is consistent with a rapid-equilibrium, random mechanism (22). For GST(atrazine) activity measured in the purified GST dimer fraction from resistant and susceptible biotypes, the GSatrazine conjugate exhibited competitive inhibition when GSH or atrazine was the varied substrate at subsaturating concentrations of the fixed substrate (Figs. 8 and 9). These results eliminate a steady-state, ordered mechanism which would

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FIG. 6. Double-reciprocal plots of initial-velocity data for GST(atrazine) activity from leaves of the atrazinesusceptible biotype. (A) atrazine concentrations (mM⫺1) varied at fixed concentrations of GSH: 0.5 mM, 䡬; 1.0 mM, 䡺; 2.0 mM, 䉭; 4.0 mM, 䉮. (B) GSH concentrations (mM⫺1) varied at fixed concentrations of atrazine: 0.08 mM, 䡬; 0.16 mM, 䡺; 0.32 mM, 䉭; 0.64 mM, 䉮. Insets are replots of y-intercepts versus the reciprocal of the fixed substrate concentration. ␯ ⫽ nmol h⫺1 mL⫺1.

have resulted in mixed-type, slope/intercept plots for both varied substrates (22). The doublereciprocal plots from the product inhibition analysis (Figs. 8 and 9) are those predicted by competitive inhibition and are consistent with a random, rapid-equilibrium mechanism.

The kinetic constants generated by fitting the initial-velocity data to the random, rapid-equilibrium model are summarized in Table 3. The dissociation constants for GSH and atrazine determined for GST(atrazine) activity for the resistant biotype are within the standard error

FIG. 7. Double-reciprocal plots of initial-velocity data of GST(atrazine) activity from leaves of the atrazineresistant biotype. (A) atrazine concentrations (mM⫺1) varied at fixed concentrations of GSH: 0.5 mM, 䡬; 1.0 mM, 䡺; 2.0 mM, 䉭; 4.0 mM, 䉮. (B) GSH concentrations (mM⫺1) varied at fixed concentrations of atrazine: 0.08 mM, 䡬; 0.16 mM, 䡺; 0.32 mM, 䉭; 0.64 mM, 䉮. Insets are replots of y-intercepts versus the reciprocal of the fixed substrate concentration. ␯ ⫽ nmol h⫺1 mL⫺1.

Abutilon GLUTATHIONE S-TRANSFERASES

43

FIG. 8. Reciprocal plots of GS-atrazine inhibition of initial-velocity GST (atrazine) activity from leaves of the atrazine-susceptible biotype. (A) GSH concentration fixed at 1 mM with atrazine concentrations varied at fixed GSatrazine concentrations; (B) atrazine concentration fixed at 0.2 mM with GSH concentrations varied at fixed GS-atrazine concentrations. GS-atrazine concentrations: 0 mM, 䡬; 0.025 mM, ●; 0.050 mM, 䡺; 0.100 mM, 䡲; 0.200 mM, 䉭.

FIG. 9. Reciprocal plots of GS-atrazine inhibition of initial-velocity GST(atrazine) activity from leaves of the atrazine-resistant biotype. (A) GSH concentration fixed at 1 mM with atrazine concentration varied at fixed GS-atrazine concentrations; (B) atrazine concentration fixed at 0.2 mM with GSH concentrations varied at fixed GS-atrazine concentrations. GS-atrazine concentrations: 0 mM, 䡬; 0.05 mM, ●; 0.100 mM, 䡺; 0.200 mM, 䡲; 0. 400 mM, 䉭.

of the constants determined for the susceptible biotype. The ␣ values of approximately 1.0 for GST(atrazine) activity for both resistant and susceptible biotypes indicate little or no effect of binding of one substrate on the subsequent binding of the second substrate. Although there are no significant differences in Km values for GSH and atrazine between the two biotypes, the kcat for GST(atrazine) is approximately 3-fold

greater for the resistant biotype compared to the susceptible biotype. The Ki values for GSatrazine versus GSH or atrazine are 1.9- and 1.5fold greater for GST(atrazine) in the resistant biotype compared to the susceptible biotype. DISCUSSION

The results of this study are in agreement with the earlier study by Anderson and Gronwald (4)

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TABLE 3 Kinetic Constants for GST (Atrazine) Activity Measured in a Purified GST Protein Fraction from Leaves of AtrazineSusceptible and -Resistant Velvetleaf Biotypes (Means ⫾ SE) Biotype

Kinetic constant

Susceptible

KA (GSH)a KB (atrazine)a ␣b c k cat K a,d i K a,e i

0.83 ⫾ 0.25 0.19 ⫾ 0.06 1.2 ⫾ 0.5 316 ⫾ 20 0.088 ⫾ 0.010 0.095 ⫾ 0.006

Resistant 1.49 ⫾ 0.40 0.27 ⫾ 0.08 1.0 ⫾ 0.5 1036 ⫾ 88 0.169 ⫾ 0.009 0.142 ⫾ 0.017

a

mM. ␣, parameter describing the effect of binding one substrate on the affinity of the enzyme for the second substrate; ␣KA ⫽ KM for GSH; ␣KB ⫽ KM for atrazine. c h⫺1. Calculation based on average Mr ⫽ 2.6 ⫻ 104. d Inhibition constant for GS-atrazine with GSH as the varied substrate. e Inhibition constant for GS-atrazine with atrazine as the varied substrate. b

which concluded that enhanced atrazine metabolism in the atrazine-resistant biotype was not due to modified Km values for the substrates atrazine and GSH, but rather was due to increased Vmax for GST(atrazine) activity. The 3-fold increase in Vmax measured by Anderson and Gronwald (4) in a crude extract is in close agreement with the 3.3-fold increase in kcat for atrazine activity measured in the purified GST fraction obtained in this study. Anderson and Gronwald (4) suggested that the increase in Vmax measured in relatively crude extracts from the resistant biotype was due to overexpression of GST(atrazine) protein. However, the results of this study indicate that is not the case. There is no significant difference in total GST protein (dimer plus monomer) between resistant and susceptible biotypes. Furthermore, there appear to be no qualitative or quantitative differences in GST subunits or isoforms in resistant and susceptible biotypes as indicated by SDS-PAGE and native gels of GST protein, respectively. Atrazine resistance in the velvetleaf biotype investigated in this study is inherited as a single nuclear gene exhibiting partial dominance (1). The results of this and previous studies (3,4) are

consistent with the hypothesis that resistance is due to the mutation of a nuclear gene encoding a GST subunit that exhibits activity with atrazine. Presumably, the mutation has resulted in the substitution of an amino acid residue in the catalytic site of the resistant enzyme which in turn has led to an enhanced kcat for GST (atrazine) activity and an increase in Ki values for GS-atrazine. The kinetic analysis in this study was performed on a purified GST fraction that contained three major and two minor GST isoforms. The validity of the kinetic analysis rests on the following assumptions: (1) both resistant and suceptible biotypes have one gene encoding a GST subunit that exhibits activity with atrazine, and (2) the GST(atrazine) subunit exhibits catalytic independence if present as a component of a heterodimer. Support for the first assumption is our finding that the reciprocal plots from both resistant and susceptible biotypes were linear (Figs. 6 and 7) and the velocity curves hyperbolic (data not shown). If multiple GST subunits having differing activities with atrazine were present in our purified GST fraction, the kinetic analysis would have yielded curved reciprocal plots and biphasic velocity curves (22). In support of the second assumption are reports indicating that the subunits of mammalian GST heterodimers are catalytically independent (30,31). Furthermore, the results of kinetic analysis of the sorghum GST B1/B2 heterodimer were consistent with catalytic independence of GST subunits (27). For mammalian GSTs, most evidence suggests a kinetic model involving a sequential, random, Bi Bi mechanism (32–39). However, there is lack of agreement as to whether the kinetic mechanism for mammalian GSTs is steady-state or rapid-equilibrium. GST 3-3 and GST 2-2 from rat liver exhibit a random, steadystate mechanism (32,39). In contrast, other GSTs have been described as best fitting a random, rapid-equilibrium model. These include GST 1-1 from rat liver (33), human placental GST P1-1 (formerly GST ␲) (34,37,38), mouse GST YfYf (36), and a GST from bovine brain (35). It has been suggested that the kinetic mechanism

Abutilon GLUTATHIONE S-TRANSFERASES

for mammalian GSTs may be isozyme-dependent (34). We previously reported that a sequential, random, rapid-equilibrium, Bi Bi model provided a good fit to the kinetic data for GST A1/A1 isolated from sorghum (27). Although this model provided a good fit to the data, definitive experiments involving product inhibition, which are needed to confirm this model, were not conducted with GST A1/A1. The kinetic analysis of GST(atrazine) activity from velvetleaf reported herein also supports a sequential, random, rapid-equilibrium Bi Bi model. In this case, we confirmed this kinetic mechanism by conducting product inhibition studies. The demonstration that the GS-atrazine conjugate is competitive with either substrate (GSH, atrazine) under nonsaturating conditions and is not inhibitory when the concentrations of both substrates are saturating is consistent with the random, rapid-equilibrium kinetic model (22). Initial-velocity studies indicated that kcat for GST(atrazine) activity was approximately 3-fold greater for the resistant biotype. For bireactant enzymes that exhibit sequential, random, rapidequilibrium kinetics, such as GST(atrazine), the rate-limiting step is the conversion of the ternary complex EAB to EPQ where E ⫽ enzyme, A and B ⫽ substrates, and P and Q ⫽ products. Substrate binding and product release are considered to be very rapid and not rate-limiting (22). It is probable that the mutation of the GST(atrazine) subunit in the resistant biotype has accelerated the conversion of the enzymeGSH-atrazine ternary complex to the enzymeGS-atrazine-Cl⫺ ternary complex. Site-directed mutagenesis studies of GSTs that exhibit random, rapid-equilibrium kinetics illustrate the importance of certain residues in the electrophilic-binding site on kcat. For human placental GST P1-1, the effect of site-directed mutagenesis on kcat was substrate-dependent (38). A Y108F mutation in the electrophilic-binding site of GST P1-1 enhanced kcat for NBD-Cl but also increased the Km for this susbtrate. It was postulated that enhanced kcat for NBD-Cl in the Y108F mutant was due to the effect of this mutation on the structural transition of the ternary complex

45

which appears to be rate-limiting with this substrate. In constrast, the same mutation resulted in a 15-fold decrease in kcat with ethacrynic acid as substrate with no change in Km. The Y108F mutation had no effect on kinetic parameters when CDNB was used as substrate. Based on in vitro experiments, it appears that enhanced atrazine metabolism in leaves of the resistant biotype is due to accelerated kcat for GST(atrazine). However, product inhibition, which is not a factor in the in vitro determinations of kcat, needs to be considered in vivo. Kinetic analysis indicated that GS-atrazine has a higher affinity for free enzyme than the substrates GSH and atrazine. For example, in the case of the susceptible biotype, the Ki values for GS-atrazine are approximately 10-fold and 2.5fold lower than the Km values for GSH and atrazine, respectively. The Ki values for GS-atrazine versus GSH or atrazine are 1.9- and 1.5-fold greater for GST(atrazine) in the resistant biotype. Hence, GST(atrazine) activity in leaves of the resistant biotype may be less subject to product inhibition compared to the susceptible biotype. However, the relative importance of product inhibition in vivo is dependent on the concentrations of the substrates GSH and atrazine. If either substrate is saturating, product inhibition will not occur. Product inhibition is also dependent on the level of GS-atrazine which could be influenced by degradation and/or compartmentation. In contrast to sorghum, where GS-atrazine is metabolized (40), there does not appear to be significant degradation of this product in velvetleaf (3). Whether GS-atrazine is compartmentalized in the vacuole of velvetleaf has not been examined. In barley mesophyll cells, the tonoplast has a transporter that moves the GSH conjugate of simazine, a close analog of atrazine, into the vacuole (41). It appears that most GST isozymes found in velvetleaf are glycoyslated as indicated by binding of conA-biotin. Previously, we reported that two sorghum GST isozymes (GST A1/A1 and GST B1/B2) were glycosylated (27). As with the subunits of sorghum GST A1/A1 and B1/

46

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B2, there were differences in the level of glycosylation of GST subunits in velvetleaf. Velvetleaf GSTs are also similar to sorghum GST A1/A1 and GST B1/B2 in that they do not appear to be heavily glycosylated since glycosylation was not detectable by the PAS reagent which is less sensitive than detection by conA-biotin. ConA-biotin, which contains six molecules of biotin per molecule of conA, allows for low levels of glycosylation to be visualized. Other studies have indicated the greater sensitivity of conA for detecting protein glycosylation compared to the PAS reagent (42,43). There are relatively few reports of post-translational modification of GSTs. Phosphorylation and methylation of mammalian GSTs have been catalyzed in vitro. Rat liver cytosolic GSTs were phosphorylated by a Ca2⫹-phosopholipiddependent protein kinase from rabbit brain (44). Calmodulin-stimulated methylation of rat cytosolic GSTs in vitro has been reported (45). There is one report indicating that certain GSTs isolated from mammals are glycosylated. Human GST ␲ and rat GST Yp were detected as glycoproteins with fluorescein isothiocyanate-conA, a fluorescent conA conjugate (43). As with velvetleaf GSTs, glycosylation of GST ␲ and GST Yp could not be detected with a visualization technique that utilized periodate oxidation. The function of glycosylation of mammalian and plant GSTs is not known. Previous studies have indicated that protein glycosylation may play a role in protein folding and stabilization, reducing susceptibility to proteases, compartmentation, and recognition phenomena (46). The presence of a putative GST monomer in crude extracts from velvetleaf leaves and in the eluent from the DEAE column was unexpected. Although found in both resistant and susceptible biotypes, only in the resistant biotype did the monomer exhibit GST(atrazine) activity. However, GST(atrazine) activity of the monomeric GST from the resistant biotype was labile and rapidly lost when attempts were made at further purification. The labile nature of the monomer was not surprising because dimeric quartenary structure is required by GSTs for the maintance of a catalytically active site (47,48). It is possible

that the GST monomers found in leaves of velvetleaf may be an artifact generated during extraction. However, it was not generated on the DEAE column because it was present in crude extracts. Alternatively, the putative monomers may represent a degradation product generated in vivo during enzyme turnover. There are no previous reports of GST monomers in plants. However, there is one report of monomeric GSTs in mammalian tissues. Monomers of GST-␲ were secreted into human plasma by platelets and tumor cells via an energy-dependent process (49). The secreted monomers exhibited no activity and their function was not determined. GSTs have been implicated in acquired insecticide resistance in insects (50–56) and in resistance of mammalian tumor and nontumor cells to various drugs and chemotherapeutic agents (57–61). In most cases, resistance is associated with overexpression or amplification of GSTs that detoxify the xenobiotic. However, in the case of the atrazine-resistant velvetleaf biotype investigated herein enhanced atrazine detoxification is not due to overexpression of GSTs. Nor is it due to an altered pattern of GST isozyme expression based on results obtained from SDSPAGE and native IEF gels. Instead, resistance is the result of enhanced kcat for GST(atrazine) activity. The potential for mutations at the electrophilic substrate binding site of GSTs to result in xenobiotic resistance due to enhanced kcat was demonstrated by Gulick and Fahl (62). Rat GSTs that had been randomly mutated in the electrophilic substrate binding site were expressed in E. coli and the cells subjected to selection pressure by the alkylating agent mechlorethamine. The majority of mechlorethamine-resistant cells contained mutant GSTs that exhibited enhanced kcat for mechlorethamine but little or no change in Km for this substrate. The authors postulated that mutations in the electrophile binding site of GSTs that result in enhanced kcat for a particular xenobiotic may be an important mechanism in the development of GST-mediated resistance. Whether the enhanced kcat for GST (atrazine) activity in the atrazine-resistant velvetleaf biotype is due to a mutation in the electrophile binding site of the GST(atrazine) subunit is not

Abutilon GLUTATHIONE S-TRANSFERASES

known. Further research is needed to clone the gene encoding the GST(atrazine) subunit and to determine whether a mutation has occurred in this region. Recently, atrazine resistance due to enhanced GSH conjugation has been reported for a velvetleaf biotype in Wisconsin (5). It would be of interest to determine whether a similar modification in the kinetics of GST-mediated conjugation of atrazine has occurred in the resistant Wisconsin biotype. ACKNOWLEDGMENTS We thank Novartis (Greensboro, NC) for providing the radiolabeled and technical grade atrazine used in this study. Cooperative investigation of the USDA-Agricultural Research Service and the Minnesota Agricultural Experiment Station. Paper No. 97-1-13-0022, Scientific Journal Series, Minnesota Agricultural Experiment Station, St. Paul, Minnesota 55108. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product and the use of the name by USDA implies no approval of the product to the exclusion of others that may be suitable.

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