Refolding and Characterization of Rat Liver Methionine Adenosyltransferase from Escherichia coli Inclusion Bodies

Protein Expression and Purification 19, 219 –226 (2000) doi:10.1006/prep.2000.1235, available online at http://www.idealibrary.com on

Refolding and Characterization of Rat Liver Methionine Adenosyltransferase from Escherichia coli Inclusion Bodies M. Carmen Lo´pez-Vara,* Marı´a Gasset,† and Marı´a A. Pajares* ,1 *Instituto de Investigaciones Biome´dicas “Alberto Sols” (CSIC), Arturo Duperier 4, 28029 Madrid, Spain; and †Instituto de Quı´mica-Fı´sica “Rocasolano” (CSIC), Serrano 119, 28006 Madrid, Spain

Received December 3, 1999, and in revised form February 29, 2000

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the major methyl donor for transmethylation reactions. Attempts to perform structural studies using rat liver MAT have met with problems because the protein purified from cellular extracts is heterogeneous. Overexpression of the enzyme in Escherichia coli rendered most of the protein as inclusion bodies. These aggregates were purified by specific washes using urea and Triton X-100 and used for refolding. Maximal activity was obtained when chaotropic solubilization included the structural cation Mg 2ⴙ, the protein concentration was kept below 0.1 mg/ml, and denaturant removal was carried out in a two-step process, namely, a fast dilution followed by dialysis in the presence of 10 mM DTT or GSH/GSSG redox buffers. Refolding by this procedure generated the oligomeric forms, MAT I and III, which were basically indistinguishable from the purified rat liver forms in secondary structure and catalytic properties. © 2000 Academic Press

Many of essential functions for the cell depend on methylation reactions that use S-adenosylmethionine (AdoMet) 2 as the methyl donor. These reactions include DNA methylation and synthesis of phospholipids, neurotransmitters, and many other small molecules (1). Alterations in AdoMet synthesis, and, hence, in its levels, have been observed in several diseases (2). These findings focused attention on methionine adeno1 To whom correspondence should be addressed. Fax: 34-915854587. E-mail: [email protected]. 2 Abbreviations used: AdoMet, S-adenosylmethionine; MAT, methionine adenosyltransferase; DMSO, dimethyl sulfoxide; PMSF, phenylmethanesulfonyl fluoride; DTT, dithiothreitol; GSH, glutathione; GSSG, glutathione disulfide; IPTG, isopropyl ␤-D-thiogalactopyroroside; CD, circular dichroism.

1046-5928/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

syltransferase (MAT) (EC 2.5.1.6), the enzyme that catalyzes the production of AdoMet from methionine and ATP in a singular reaction that requires Mg 2⫹ and K ⫹ ions (3,4). In mammals, 85% of the transmethylation reactions take place in the liver (1), where about 48% of the ingested methionine is metabolized to produce, in humans, 6 – 8 g of AdoMet daily (5,6). For this purpose, the liver contains a specific MAT isoenzyme. This enzyme is made of a single amino acid chain of 396 residues containing 10 cysteines (7,8) and appears as a homotetramer (MAT I) and a homodimer (MAT III, consensus nomenclature for this field) (9,10). The oligomeric assemblies differ in their affinities for methionine. MAT I is 10-fold more active than MAT III under physiological concentrations of methionine (60 ␮M). MAT III is specifically activated by dimethyl sulfoxide (DMSO) (9,11). The presence of specific hepatic MAT forms has led to speculations about their role in the regulation of liver methionine metabolism (12). MAT I with a higher affinity and more activity at physiological concentrations of methionine is thought to be active under basal levels of the amino acid. However, when methionine levels increase during feeding, a MAT form (MAT III), with higher capacity but lower affinity, is thought to be necessary for fast serum clearance. Therefore, changes in the MAT I/III ratio will affect methionine clearance rates and will be under strict control. The conditions altering the MAT I/III ratio have been studied both in vitro and in vivo. Changes in enzyme activity have been correlated to alterations in the ratio MAT I/III in several in vitro experiments (13–16). In vivo, decreases in MAT activity have been observed in animal models where reductions in the GSH concentration were produced (17,18). Similarly, reductions in MAT activity, AdoMet levels, and a change in the ratio MAT I/III 219

220

´ PEZ-VARA, GASSET, AND PAJARES LO

have been obtained in samples of patients with alcohol liver cirrhosis (2). However, the data available have not provided exact knowledge of the mechanisms controlling the change. To establish the structural basis of the complex regulation profile of this enzyme we first cloned (8) and overexpressed the hepatic rat MAT in a heterologous system, Escherichia coli (10,19). This system, however, produces a considerable amount of MAT as insoluble aggregates (10,19). To overcome this problem we have refolded and purified recombinant rat liver MAT from E. coli inclusion bodies, which can be used for structural and regulatory studies. In addition, recombinant MAT offers a promising alternative for the biotechnological production of AdoMet. MATERIALS AND METHODS

Materials. Methionine, ATP, benzamidine, phenylmethanesulfonyl fluoride (PMSF), pepstatin A, aprotinin, leupeptin, antipain, dithiothreitol (DTT), ampicillin, glutathione (GSH), glutathione disulfide (GSSG), and the molecular mass standards for gel filtration chromatography were products from Sigma Chemical Company (St. Louis, MO). [2- 3H]ATP (20 Ci/mmol) was supplied by Amersham International (Little Chalfont, Buckinghamshire, UK). Isopropyl ␤-D-thiogalactopyranoside (IPTG) was a product of Ambion (Austin, TX). DEAE–Sephacel, phenyl Sepharose CL-4B, Superose 12 HR 10/30, and isoelectric focusing reagents were purchased from Pharmacia LKB (Uppsala, Sweden). Optiphase HiSafe 3 scintillation fluid was obtained from E & G Wallac (Milton Keynes, UK). Cation exchanger AG-50W-X4, Bio-Gel A 1.5 m, goat anti-rabbit IgG– horseradish peroxidase, Bio-Rad protein assay kit, and the electrophoresis reagents were from BioRad (Richmond, CA). Ultrafiltration membranes YM-30 were purchased from Amicon Corp. (Beverly, MA). Chemiluminescence Renaissance reagents were obtained from DuPont New England Nuclear (Boston, MA). Urea, DMSO, and Triton X-100 were purchased from Merck (Darmstadt, Germany). The rest of the buffers and reagents were of the best quality commercially available. Expression of rat liver MAT and purification from E. coli inclusion bodies. Competent E. coli BL21(DE3) cells were transformed with the plasmid pSSRL-T7N (10) and 500-ml cultures were prepared as described previously after induction for 3 h (19). Cells were harvested by centrifugation, washed with water, and stored at ⫺70°C until use. Cell pellets were disrupted by sonication at 4°C in a Branson 250 sonifier (15 pulses of 30 s at 30-s intervals, output power level 20) in 50 ml of 50 mM Tris/HCl, pH 8.0, containing 0.5 M NaCl, 0.1% (v/v) 2-mercaptoethanol, and protease inhibitors (2 ␮g/ml aprotinin, 1 ␮g/ml pepstatin A, 0.5

␮g/ml leupeptin, 2.5 ␮g/ml antipain, 0.1 mM benzamidine, 0.1 mM PMSF). Soluble and insoluble fractions (including inclusion bodies, 1–1.5 g wet wt) were separated by centrifugation for 15 min at 13,000g. All extraction and the purification procedures were carried out at 4°C. Pellets of the insoluble fraction were suspended in 12.5 ml of 10 mM Hepes/Na, pH 7.5, containing 10 mM MgSO 4 and 1 mM EDTA (buffer A) and disrupted by sonication (six pulses of 10 s with 10-s intervals, output power level 20). The solution was centrifuged for 30 min at 4°C at 48,000g and the supernatant was discarded. The pellet was washed twice by resuspension in 12.5 ml of 100 mM Tris/HCl, pH 7.0, containing 5 mM EDTA, 5 mM DTT, 4 M urea, 5% Triton X-100, and 0.1 mM benzamidine, and centrifuged for 30 min at 4°C at 48,000g. A last wash with the same buffer devoid of urea and Triton X-100 was performed, and the preparation was stored at ⫺20°C until use. MAT purity was assessed after solubilization in 50 mM Tris/ HCl, pH 8, containing 8M urea by SDS–PAGE, IEF, immunoblotting, and N-terminal sequencing. Refolding of rat liver MAT. The washed pellet was solubilized in 50 mM Tris/HCl, pH 8.0 (buffer B), containing 8 M urea. Protein concentrations (0.3–18 mg/ ml), incubation time (0 to 24 h), temperature (37°C, 10°C, and room temperature), and the presence of several additives in buffer B (0 –200 mM MgSO 4, 0 –300 mM KCl, 0 –100 mM DTT, 0 –5 mM Met, 0 –5 mM ATP, and their combinations) were varied for optimization studies. Refolding was then performed by direct dialysis or by dilution to lower urea concentrations (8 –1 M) before the dialysis step. Dilution and dialysis were performed using buffer B, but in the dialysis step several additives were tested. The additives included DTT (0 –50 mM), GSH (0 –50 mM), 2-mercaptoethanol (0 –50 mM), MgSO 4 (0 –100 mM), and GSH/GSSG redox buffers (2:1, 5:1, and 10:1). Dialysis was also performed at several pHs, ranging from 6.0 to 8.5. Dialyzed samples were clarified by centrifugation at 48,000g for 30 min before any determination was carried out. Purification of refolded rat liver MAT. MAT III refolded in the presence of DTT was purified on a DEAE– Sephacel column (2.5 ⫻ 12 cm) equilibrated in 50 mM Tris/HCl, pH 8, 10 mM MgSO 4 (buffer C), that was washed with 3 bed vol of buffer C and eluted with a KCl gradient from 0 to 500 mM in buffer C. Isolation of MAT forms refolded in the presence of GSH/GSSG required an additional step on a phenyl Sepharose CL-4B column (2.5 ⫻ 10 cm) equilibrated in buffer C. The column was washed with 3 bed vol of equilibration buffer, followed by 2 bed vol of buffer C containing 50% (v/v) DMSO. Under these conditions MAT I and III refolded in the presence of GSH/GSSG were obtained. When required, buffer exchanges were performed by

221

METHIONINE ADENOSYLTRANSFERASE REFOLDING

FIG. 1. Characterization by SDS–PAGE, immunoblotting, and isoelectrofocusing of the purified DTT-refolded MAT. Inclusion bodies were obtained and washed as described under Materials and Methods. MAT refolding was then carried out in the presence of 10 mM DTT and the protein was purified. Samples of the purified MAT (10 ␮g) were used for SDS–PAGE, IEF, and immunoblotting. (A) Coomasie blue staining of the SDS–PAGE gel. The molecular mass of the markers is shown on the left, and the calculated molecular mass of the band appears on the right. Similar results were observed when the electrophoresis was performed in the absence of reducing agents and when silver staining was carried out. (B) Immunoblot obtained using an anti-rat liver MAT antiserum. Again the molecular mass calculated for the band is stated on the left. (C) IEF gel of the sample depicting the pI of the markers on the right and the calculated pI for MAT on the left side of the lane. Similar results were obtained when using the proteins refolded in the presence of GSH/GSSG.

gel filtration on a Bio-Gel A 1.5-m column (1.5 ⫻ 90 cm) using concentrated protein samples. Purification of rat liver MAT. Rat liver MAT I and III were purified as described previously (15), except that thiopropyl Sepharose chromatography was omitted. MAT activity assay. MAT activity was measured essentially as described previously (20), using protein

concentrations of 0.05 mg/ml. For MAT I and III differentiation, the activity was measured using 60 ␮M methionine, either in the presence or in the absence of 10% (v/v) DMSO. Optimum pH of refolded MAT forms was determined at saturating concentrations of the substrates using reaction mixtures in the 6.5 to 10 pH range. Kinetic parameters of the refolded recombinant rat liver MAT. Activity assays to determine the kinetic parameters of the refolded protein forms were performed as described above, but using different concentrations of methionine, ATP, KCl, and MgCl 2 in the reaction mixture. Methionine and ATP kinetics were carried out as described previously (15). As for Mg 2⫹ and K ⫹ kinetics, protein samples preequilibrated in cation-depleted buffers and reaction mixtures containing 5 mM methionine and 5 mM ATP were used. Mg 2⫹ kinetics were performed at 0 –36 mM in the presence of 250 mM KCl, whereas K ⫹ kinetics were performed at 0 –360 mM in the presence of 9 mM MgCl 2. Chromatographic characterization of refolded MAT forms. Samples of refolded MAT (100 ␮l containing a minimum of 20 ␮g) were injected on a Superose 12 HR 10/30 gel filtration column connected to an Advanced Protein Purification System (Waters). Equilibration and elution were performed using buffer C containing 150 mM KCl, 1 mM EDTA at a flow rate of 0.3 ml/min, and 210-␮l fractions were collected. MAT activity and absorbance at 280 nm were measured in each fraction. Circular dichroism. Far-UV circular dichroism (CD) spectra of MAT forms were recorded in a Jasco J-720 spectropolarimeter at 25°C (21), using samples of about 0.1 mg/ml protein concentration and 0.1-cmpathlength cuvettes. After baseline subtraction the observed ellipticities were converted to mean residue ellipticities (Q mrw) on the basis of a mean molecular mass

TABLE 1 Influence of Different Additives in the Dialysis Buffer on the Recovery of MAT Activity

Additives None 10 mM 10 mM 10 mM 10 mM 10 mM 10 mM 10 mM

2-mercaptoethanol GSH DTT DTT ⫾ 10 mM MgSO 4 a GSH/1 mM GSSG GSH/1 mM GSSG a GSH/1 mM GSSG ⫹ 10 mM MgSO 4 a

Urea-denatured MAT (nmol/min/mg)

Urea-denatured MAT ⫹ 75 mM MgSO 4 (nmol/min/mg)

2.7 ⫾ 0.2 3.5 ⫾ 0.1 5.8 ⫾ 0.4 17.3 ⫾ 0.6 30.0 ⫾ 0.7 1.5 ⫾ 0.2 ND ND

ND ND 14.6 ⫾ 1.5 39.3 ⫾ 4.7 95.0 ⫾ 8.5 8.8 ⫾ 0.7 57.8 ⫾ 1.5 81.0 ⫾ 2.6

Note. Urea-denatured MAT (0.1 mg/ml) was incubated for 2 h at 10°C. Refolding was then carried out by the one- or two-step procedure as described under Materials and Methods. In both cases the effect of several additives in the dialysis buffer was analyzed by measuring MAT activity in the presence of 5 mM methionine. The results shown are the mean ⫾ SD of a typical experiment done in triplicate. a Values obtained by the two-step refolding procedure, including dilution to ⬍2 M urea.

222

´ PEZ-VARA, GASSET, AND PAJARES LO

ical calculation of the pI was performed using a method previously developed (25). N-terminal amino acid sequencing. For N-terminal sequencing, proteins (10 –20 ␮g) resolved on 10% SDS– PAGE gels were electrotransferred to Immobilon-P membranes using 10 mM Caps, pH 11, containing 10% (v/v) methanol, and stained using Ponceau S and the band was excised. Sequencing was performed on an Applied Biosystems Procise sequencer. Protein concentration determination. The protein concentration of the samples was measured using the Bio-Rad protein assay kit (26) and spectrophotometrically using a calculated molar extinction coefficient at 280 nm of 41934 M ⫺1 cm ⫺1 in 8 M urea. RESULTS AND DISCUSSION

Rat liver MAT was expressed in E. coli using the plasmid pSSRL-T7N. After IPTG induction, however,

FIG. 2. Two-step refolding procedure. Urea-denatured MAT at 0.1 mg/ml was incubated for 2 h at 10°C, in the presence (filled circles) or absence (open circles) of 75 mM MgSO 4. Refolding was then carried out by a fast step (denaturant dilution) followed by dialysis. MAT activity was then measured in triplicate in the presence of 5 mM methionine. Concentrations of denaturant upon dilution are shown in the figure. Dialysis was carried out in the presence of 10 mM DTT (filled and open circles). The results shown in the figure are the mean ⫾ SD of a typical experiment carried out in triplicate. Similar results were obtained when refolding in the presence of GSH/GSSG was performed.

per residue of 110 Da. Secondary structure composition was calculated using the Convex Constraint Analysis (CCA) with the original set of reference proteins (22). SDS–PAGE, immunoblotting, and isoelectric focusing. Denaturing gel electrophoresis was performed on 10% SDS–PAGE gels under reducing conditions using the buffer system described previously (23). Samples containing 10 –20 ␮g of protein or 20 ␮l of the resuspended lyophilized samples were loaded per lane. Immunoblotting analysis was carried out as described by Mingorance et al. (19). Protein bands were visualized by chemiluminescence using DuPont New England Renaissance reagents. Isoelectric focusing was performed using 10% acrylamide gels and a pH gradient from 3.5–10.0 (24). Standards for pI determination were: amyloglucosidase (3.50), soybean trypsin inhibitor (4.55), ␤-lactoglobulin A (5.20), bovine carbonic anhydrase B (5.85), human carbonic anhydrase B (6.55), myoglobin-acidic band (6.85), myoglobin-basic band (7.35), lentil lectin-acidic band (8.15), lentil lectin-middle band (8.45), lentil lectin-basic band (8.65), and trypsinogen (9.30). Theoret-

FIG. 3. Gel filtration chromatography of refolded MAT samples. Urea-denatured MAT in the presence of 75 mM MgSO 4 was used for the two-step refolding procedure. DTT or GSH/GSSG was included in the dialysis mixture. Gel filtration chromatography was then carried out as described under Materials and Methods using a Superose 12 HR column. Fractions of 210 ␮l were collected and used for measuring MAT activity. (A) The profile of purified rat liver MAT I (open circles) and MAT III (filled circles). The chromatograms of MAT refolded in the presence of DTT (B) and GSH/GSSG (C) are also shown in the figure. In each case 20 –50 ␮g of protein was injected. The elution volume of the markers was 8.95 ml for dextran blue (2000 kDa); 12.4 ml for ␤-amylase (200 kDa); 13.6 ml for alcohol dehydrogenase (150 kDa); 14.8 ml for bovine serum albumin (66.2 kDa); and 16 ml for carbonic anhydrase (29 kDa).

223

METHIONINE ADENOSYLTRANSFERASE REFOLDING

TABLE 2 Refolding and Purification of MAT I and MAT III

Urea Dialysis ⫹ DTT DEAE–Sephacel Dialysis ⫹ GSH/GSSG Phenyl–Sepharose MAT I Phenyl–Sepharose MAT III

Total protein (mg)

Total enzyme units

Specific activity (nmol/min/mg)

Yield (%)

18.7 16.6 11.3 16.5 4.4 10.3

0 1576 949 1337 434 684

⬍0.00001 95 84 81 99 67

100 88 60 88 23 55

Note. Washed inclusion bodies were used for refolding after solubilization in 50 mM Tris/HCl, pH 8, 75 mM MgSO 4, 8 M urea for 2 h at 10°C. The samples were diluted to ⬍2 M urea and dialyzed against 50 mM Tris/HCl, pH 8, 10 mM MgSO 4, including either DTT or GSH/GSSG. MAT forms produced were purified and used for the rest of the studies. The table shows the results of a typical purification.

the enzyme appeared mostly in the inclusion bodies (10). Initial attempts to improve the yield of soluble protein were unsuccessful, and hence an alternative protocol for obtaining active MAT I/III was established using as a source the inactive aggregates. Successive washes of these inclusion bodies with 4 M urea and 5% Triton X-100 removed the extraneous proteins, producing an essentially homogeneous MAT preparation, as judged by both SDS–PAGE gels and immunoblotting (Fig. 1). The calculated M r of the monomer (48,000) and the pI of 5.9 provided by IEF analysis agreed with the values obtained for the purified rat liver enzyme and with the values predicted from the sequence (Fig. 1) (9,15,19,25). Furthermore, N-terminal sequencing resulted in MNGPVDG-, as expected from the MAT sequence (7,8). Together all these data supported fulllength integrity. Quantitative solubilization of MAT was achieved by denaturation using 8 M urea at pH 8.0 for 2 h at 10°C. Refolding was then accomplished by chaotrope removal. The determination of the best refolding condi-

tions is a process that has to be developed for each particular protein (27). The presence of 10 cysteines in rat liver MAT, the Mg 2⫹ dependence of its activity, and location of the active sites at the dimer interface (28) suggested that analysis of several factors for optimal refolding would have to be considered. Such factors include protein concentration, ionic and redox buffer requirements, and the procedure for chaotrope removal. Denaturant removal using slow and/or fast steps and the presence on refolding of reducing and/or oxidizing agents have been described to produce different results depending on the protein under study (27,29,30). This procedure is especially difficult to control when working with proteins containing a relatively large number of cysteine residues (31). The results, evaluated on the basis of the total MAT activity recovered, are shown in Table 1. Protein concentrations below 0.1 mg/ml yielded the best recoveries, probably by preventing the formation of off-pathway aggregates (32). Addition of 75 mM MgSO 4 in the solubilization/denaturation buffer increased the final

TABLE 3 Characterization of Refolded MAT Forms

M r on gel filtration chromatography Specific activity (nmol/min/mg) S 0.5 methionine (␮M) S 0.5 ATP (␮M) S 0.5 Mg 2⫹ (mM) S 0.5 K ⫹ (mM) Optimum pH DMSO activation (fold)

Rat liver purified MAT III

DTT-refolded MAT III

GSH/GSSGrefolded MAT III

Rat liver purified MAT I

GSH/GSSGrefolded MAT I

110,000 97.6 ⫾ 10.5 650 ⫾ 81 950 ⫾ 35 15 ⫾ 3.8 150 ⫾ 10 7.5–8.8 10

92,700 110 ⫾ 11 246 ⫾ 76 588 ⫾ 108 1.9 ⫾ 0.1 0.9 ⫾ 0.1 7.5–8.8 3–4

89,125 95.8 ⫾ 4.4 1120 ⫾ 150 530 ⫾ 48 4.1 ⫾ 0.4 2.2 ⫾ 0.7 8.5–10 3–5

210,000 39.2 ⫾ 2.5 125 ⫾ 42 251 ⫾ 43 12 ⫾ 3.5 7 ⫾ 2.5 7.5–8.8 None

194,900 100 ⫾ 13 743 ⫾ 81 1370 ⫾ 290 5.5 ⫾ 0.2 10.7 ⫾ 0.5 7.5–9.5 0

Note. Urea-denatured MAT in the presence of 75 mM MgSO 4 was used for refolding with the two-step procedure. Samples were refolded with DTT or GSH/GSSG and the MAT forms obtained were purified as described under Materials and Methods. The refolded forms obtained were analyzed by gel filtration chromatography on a Superose 12 HR column, and their kinetic properties were studied. Data in the table are the mean ⫾ SD of the results obtained on experiments carried out in triplicate for the refolded MATs. Data for rat liver purified MAT I and III are also included.

´ PEZ-VARA, GASSET, AND PAJARES LO

224

TABLE 4 Secondary Structure Composition of MAT Isoforms Secondary structure (%) MAT form

␣-Helix

␤-Sheet

Turns

Random

Purified rat liver MAT III DTT-refolded MAT III GSH/GSSG-refolded MAT III Purified rat liver MAT I GSH/GSSG-refolded MAT I

19 ⫾ 1.1 19 ⫾ 1.2 16 ⫾ 0.9 23 ⫾ 1.8 16 ⫾ 1.5

34 ⫾ 2.3 34 ⫾ 1.4 36 ⫾ 2.1 40 ⫾ 1.2 34 ⫾ 0.8

20 ⫾ 0.8 20 ⫾ 0.3 26 ⫾ 0.3 18 ⫾ 1 24 ⫾ 1.2

27 ⫾ 0.5 27 ⫾ 1.7 22 ⫾ 1.3 19 ⫾ 1.8 26 ⫾ 1.5

Note. Secondary structure composition was calculated from the far-UV CD spectra using the Convex Constraint Analysis. The results are displayed as the average of three independent measurements. Conditions used for sample preparation, spectra acquisition, and analysis are explained under Materials and Methods.

activity obtained, but DTT, KCl, ATP, or methionine were without effect. Moreover, the dilution of denaturant to ⬍2 M followed by dialysis in the presence of 10 mM DTT or 10 mM GSH/1 mM GSSG and 10 mM MgSO 4 was crucial for increasing the recovery of MAT activity (Table 1 and Fig. 2). A number of reports on the folding of eukaryotic proteins have shown that the fastest process occurs in a thiol/disulfide redox buffer with an optimal concentration of both agents, in our case GSH and GSSG. Normally thiol/disulfide ratios of 5–10 with thiol concentrations in the 1–5 mM range are recommended (33), but in our case the best results were obtained at 10 mM GSH at a GSH/GSSG ratio of 10:1. Moreover, in the presence of this redox buffer higher MAT activity was detected when 10 mM MgSO 4 was also included in the dialysis (Table 1). Based on these results the system of choice for further studies was the two-step refolding procedure. In order to determine which MAT oligomeric form was the end-product in our system a differential method for determining MAT I or MAT III activities was used. This method takes advantage of a property shown by MAT III, its activation by DMSO at low methionine concentrations (11). DTT refolding produced low MAT activity at 60 ␮M methionine. This activity was increased in the presence of DMSO over the whole protein range tested (data not shown). Gel filtration analysis of the oligomeric assemblies produced by this refolding system showed the presence of a single peak (Fig. 3). The elution volume corresponded to a calculated M r of 92,700, as expected for MAT III (9,14,15). The GSH/GSSG refolding products displayed DMSO activation only in the presence of Mg 2⫹, and the elution profile on gel filtration chromatography exhibited a major peak with a shoulder (Fig. 3C). The elution positions corresponded to proteins of about 194,000 and 89,100 Da as expected for MAT I and III, respectively (9,14,15). Purification of the MAT forms refolded in the presence of DTT and GSH/GSSG was performed and the

results are shown on Table 2. A repetitive decrease in specific activity has been observed after DEAE–Sephacel chromatography of the MAT III refolded in the presence of DTT, in contrast to the activation detected for purified rat liver MAT after this chromatography (9). On the other hand, the presence of MAT I and III in the GSH/GSSG-refolded sample required the use of a phenyl–Sepharose chromatography for the best separation. Dimers bind to this type of column while tetramers elute in the flowthrough (9,34). The overall yield of both procedures is within the expected range for a soluble protein. The refolded proteins were structurally and functionally characterized and compared to the purified rat liver forms (Tables 3 and 4). The specific activity of the refolded forms is at least equivalent to that determined for the purified rat liver proteins (12,15). However, differences in the affinity for the substrates, methionine and ATP, are detected in all the refolded forms, as well as a decrease in the activation by DMSO of the refolded MAT III forms compared to the purified rat liver proteins (11,12,15,34). The affinity for Mg 2⫹ ions shown by the refolded forms is similar to that of the purified rat liver enzymes (12,35), while a decrease in K ⫹ activation was detected in refolded MAT III compared to the published data (12,35). These results indicated that the organization of the active site in the refolded MAT proteins must be very similar to that of the naturally occurring forms (36). Finally, the overall secondary structures of refolded and purified rat liver MAT proteins was probed by far-UV circular dichroism spectroscopy (Table 4). MAT III (refolded in the presence of DTT and GSH/GSSG and purified from rat liver) exhibit identical far-UV spectra. On the other hand, MAT I refolded in the presence of GSH/GSSG exhibit slight differences in its far-UV spectra when compared to the purified rat liver MAT I, which could be due to the presence of covalent modifications in the rat liver protein that are absent in the recombinant forms (14). Furthermore, spectral analysis showed

METHIONINE ADENOSYLTRANSFERASE REFOLDING

similar secondary structure composition for all the refolded forms, with the major element being the ␤-sheet (Table 4). The present report demonstrates the feasibility of producing biologically active MAT upon bacterial overexpression. Although the expressed protein was insoluble it could be refolded into an active conformation identical to purified rat liver MAT I/III. The availability of bacterial-derived, nonmodified MAT should aid in crystallographic and structure/function studies of this protein.

13.

14.

15.

16.

ACKNOWLEDGMENTS The authors thank Dr. L. Alvarez and S. Kesavapany for the critical reading of the manuscript and Dr. A. Martı´nez del Pozo for his help on the CD spectra. The help of the referees in improving the grammar of the manuscript is also acknowledged. M.C.L-V is a fellow of Boehringer Ingelheim Espan˜a S.A. This work was supported by grants of the Direccio´n General de Investigacio´n Cientı´fica y Te´cnica (PB 94/0087, PM 97-0064, and PB 96/0850).

17.

18.

REFERENCES 1. Cantoni, G. L. (1975) Biochemical methylations: Selected aspects. Annu. Rev. Biochem. 44, 435– 441. 2. Martı´n-Duce, A., Ortiz, P., Cabrero, C., and Mato, J.M. (1988) S-Adenosyl-L-methionine synthetase and phospholipid methyltransferase are inhibited in human cirrhosis. Hepatology (Baltimore) 8, 65– 68. 3. Cantoni, G. L. (1953) S-Adenosylmethionine: A new intermediate formed enzymatically from L-methionine and adenosinetriphosphate. J. Biol. Chem. 204, 403– 416. 4. McQueney, M. S., and Markham, G. D. (1995) Investigation of monovalent cation activation of S-adenosylmethionine synthetase using mutagenesis and uranyl inhibition. J. Biol. Chem. 270, 18277–18284. 5. Aguilar, T. S., Benevenga, N. J., and Harper, A. E. (1974) Effect of dietary methionine level on its metabolism in rats. J. Nutr. 104, 761–771. 6. Mudd, S. H., and Poole, J. R. (1975) Labile methyl balances for normal humans on various dietary regimens. Metabolism 24, 721–735. 7. Horikawa, S., Ishikawa, M., Ozasa, H., and Tsukada, K. (1989) Isolation of a cDNA encoding the rat liver S-adenosylmethionine synthetase. Eur. J. Biochem. 184, 497–501. 8. Alvarez, L., Asuncio´n, M., Corrales, F., Pajares, M. A., and Mato, J. M. (1991) Analysis of the 5⬘ non-coding region of rat liver S-adenosylmethionine synthetase mRNA and comparison of the Mr deduced from the cDNA sequence and the purified enzyme. FEBS Lett. 290, 142–146. 9. Cabrero, C., Puerta, J. L., and Alemany, S. (1987) Purification and comparison of two forms of S-adenosyl-L-methionine synthetase from rat liver. Eur. J. Biochem. 170, 299 –304. 10. Alvarez, L., Mingorance, J., Pajares, M. A., and Mato, J. M. (1994) Expression of rat liver S-adenosylmethionine synthetase in Escherichia coli results in two active oligomeric forms. Biochem. J. 301, 557–561. 11. Hoffman, J. L., and Kunz, G. L. (1977) Differential activation of rat liver methionine adenosyltransferase isozymes by dimethylsulfoxide. Biochem. Biophys. Res. Commun. 77, 1231–1236. 12. Mato, J. M., Alvarez, L., Ortiz, P., and Pajares, M. A. (1997)

19.

20.

21.

22.

23. 24.

25.

26.

27. 28.

29.

225

S-Adenosylmethionine synthesis: Molecular mechanisms and clinical implications. Pharmacol. Ther. 73, 265–280. Pajares, M. A., Corrales, F. J., Ochoa, P., and Mato, J. M. (1991) The role of cysteine 150 in the structure and activity of rat liver S-adenosyl-L-methionine synthetase. Biochem. J. 274, 225–229. Pajares, M. A., Dura´n, C., Corrales, F., and Mato, J. M. (1994) Phosphorylation by PKC of rat liver S-adenosylmethionine synthetase: Dissociation and production of a monomer. Biochem. J. 303, 949 –955. Pajares, M. A., Dura´n, C., Corrales, F., Pliego, M. M., and Mato, J. M. (1992) Modulation of rat liver S-adenosylmethionine synthetase activity by glutathione. J. Biol. Chem. 267, 17598 –17605. Martı´nez-Chantar, M. L., and Pajares, M. A. (1996) Role of thioltranferases on the modulation of rat liver S-adenosylmethionine synthetase activity by glutathione. FEBS Lett. 397, 293– 297. Corrales, F., Ochoa, P., Rivas, C., Martı´n-Lomas, M., Mato, J. M., and Pajares, M. A. (1991) Inhibition of glutathione synthesis in the liver leads to S-adenosyl-L-methionine synthetase reduction. Hepatology (Baltimore) 14, 528 –533. Corrales, F., Gime´nez, A., Alvarez, L., Caballerı´a, J., Pajares, M. A., Andreu, H., Pare´s, A., Mato, J. M., and Rode´s, J. (1992) S-Adenosylmethionine treatment prevents CCl 4-induced S-adenosylmethionine synthetase inactivation and attenuates liver injury. Hepatology (Baltimore) 16, 1022–1027. Mingorance, J., Alvarez, L., Sa´nchez-Go´ngora, E., Mato, J. M., and Pajares, M. A. (1996) Site-directed mutagenesis of rat liver S-adenosylmethionine synthetase: Identification of a cysteine residue critical for the oligomeric state. Biochem. J. 315, 761– 766. Gil, B., Pajares, M. A., Mato, J. M., and Alvarez, L. (1997) Glucocorticoid regulation of hepatic S-adenosylmethionine synthetase gene expression. Endocrinology 138, 1251–1258. Medrano, F. J., Gasset, M., Lo´pez-Zumel, C., Usobiaga, P., Garcı´a, J. L., and Mene´ndez, M. (1996) Structural characterization of the unligated and choline-bound forms of the major pneumococcal autolysin LytA amidase: Conformational transitions induced by temperature. J. Biol. Chem. 271, 29152–29161. Perczel, A., Hollosi, M., Tusnady, G., and Fasman, G. D. (1991) Convex constraint analysis: A natural deconvolution of circular dichroism curves of proteins. Protein Eng. 4, 669 – 679. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Thomas, G., and Luther, H. (1981) Transcriptional and translational control of cytoplasmic proteins after serum stimulation of quiescent Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA 78, 5712– 5716. Ribeiro, J. M., and Sillero, A. (1991) A program to calculate the isoelectric point of macromolecules. Comput. Biol. Med. 21, 131– 141. Bradford, M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilising the principle of protein– dye binding. Anal. Biochem. 72, 248 –254. Rudolph, R., and Lilie, H. (1996) In vitro folding of inclusion body proteins. FASEB J. 10, 49 –56. Takusagawa, F., Kamitori, S., and Markham, G. D. (1996) Structure and function of S-adenosylmethionine synthetase: Crystal structures of S-adenosylmethionine synthetase with ADP, BrADP and PPi at 2.8 A resolution. Biochemistry 35, 2586 –2596. Raman, B., Ramakrishna, T., and Rao, Ch. M. (1996) Refolding of denatured and denatured/reduced lysozyme at high concentrations. J. Biol. Chem. 271, 17067–17072.

226

´ PEZ-VARA, GASSET, AND PAJARES LO

30. Vandenbroeck, K., Martens, E., D’Andrea, S., and Billiau, A. (1993) Refolding and single-step purification of porcine interferon-gamma from Escherichia coli inclusion bodies. Conditions for reconstitution of dimeric IFN-gamma. Eur. J. Biochem. 215, 481– 486. 31. Pain, R. (1987) Protein folding for pleasure and for profit. Trends Biochem. Sci. 12, 309 –312. 32. Schmid, F. X. (1989) Spectral methods of characterizing protein conformation and conformational changes in “Protein Structure: A Practical Approach” (Creighton, T. E., Ed.), pp. 251–285, IRL Press, Oxford, UK. 33. Lyles, M. M., and Gilbert, H. F. (1991) Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: Dependence of the rate on the composition of the redox buffer. Biochemistry 30, 613– 619.

34. Kunz, G. L., Hoffman, J. L., Chia, C. S., and Stremel, B. (1980) Separation of rat liver methionine adenosyltransferase isozymes by hydrophobic chromatography. Arch. Biochem. Biophys. 202, 565–572. 35. Okada, G., Teraoka, H., and Tsukada, K. (1981) Multiple species of mammalian S-adenosylmethionine synthetase: Partial purification and characterization. Biochemistry 20, 934 – 940. 36. Gilbert, H. F. (1989) Thermodynamic and kinetic constraints on thiol/disulfide exchange involving glutathione redox buffers in “Glutathione Centennial: Molecular Perspectives and Clinical Implications” (Taniguchi, T., Higashi, T., Sakamoto, Y., and Meister, A., Eds.), pp.73– 87, Academic Press, New York.