Redox Forms of Human Placenta Glutathione Transferase - Tor Vergata

Vol. 266,No.

OF BIOLOGICAL CHEMISTRY THEJOURNAL 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Issue of November 15,PP. 21409-21415.1991 Printed in U.S.A.

Redox Formsof Human Placenta Glutathione Transferase* (Received for publication, April 16, 1991)

Giorgio RicciSg, Gilbert0 Del BoccioS, Alfonso PennelliS, Mario Lo Belloll, Raffaele Petruzzellill, Anna Maria Caccurill,Donatella Barrall ,and Giorgio Federicin From the $Instituteof Biochemical Sciences, University of Chieti “G. D’Annunzio,” Chieti, the VDepartment of Biology, University of Rome “Tor Vergata” and the I/Department of Biochemical Sciences, University of Rome “La Sapienza,” Rome, Italy

Human placentaglutathione transferase (EC inhibitor specificity and immunological properties (4).All the 2.5.1.18) T undergoes anoxidative inactivation which cytosolic enzymes have a dimeric structure due to a noncoleads to the formation of an inactive enzymatic form valent association of identical or different subunits with a which is homogeneous inseveral chromatographic and molecular mass of 23-28 kDa (3). Moreover, each monomer electrophoretic conditions. This process is pH depend- contains one binding site for glutathione (GSH) (G-subsite) ent, and it occurs at appreciable rate in alkaline con- and another for the hydrophobic substrate (H-subsite) (3). ditions and in the presence of metal ions. Dithiothreitol treatmentcompletelyrestoresthe active form. -SH Several studies have been performed to define the catalytic mechanism of this enzyme, but this remains obscure at the titration data and electrophoretic studies performed present; similarly, the topography of the subsites is not clarboth on the oxidized and reduced forms indicate that one intrachain disulfide is formed, probably between ified despite the primary structure of a number of isoenzymes the two faster reacting cysteinyl groups of each sub- established so far (3). At this regard one important question unit. By the use of a specific fluorescent thiol reagent is the role, if any, of the sulfhydryls present in each subunit the disulfide forming cysteines have been identifiedas of the Piclass isoenzymes. It has been recently observed that the 47th and 101th residues. The disulfide formation the covalent modification of a single cysteine residue/moncauses changes in thetertiary structure ofthis trans- omer causes a dramaticloss of activity of the homodimeric Pi ferase as appears by CD, UV, and fluorometric anal- isoenzymes such as the rat GST 7-7 (5), the human placenta yses; evidences are provided that one orboth trypto- GST-T (5, 6), the mouse GST M I1 (5), and thehorse erythphanyl residues of each subunit together with a number rocyte GST (7). This sulfhydryl is the most reactive among of tyrosyl residues are exposed to a more hydrophilic has been environment in the oxidized form. Moreover,electro- the 4 cysteine residues of each subunit,andit phoretic data indicate that the subunit of the oxidizedidentified as the 47th amino acid both in the rat 7-7 and human placentaT isoenzymes (5,6). Whether this thiol group enzyme has an apparent molecular mass lower than is important for the maintenance of a catalytically active that of the reduced transferase, thereby confirming structure or whether it is implicated in the catalytic mechastructural differences between these forms. nism remains to be clarified. Probably related to theintegrity of this residue are also the observations that oxidizing agents or disulfides cause a loss of the GSTactivity (4, Glutathione transferase (EC 2.5.1.18) (GST)’ is a family of such as Hz02 enzymes found in numerous species and in many tissues of 5, 7-10) and that a number of GST purifications were permammals (1, 2). They represent one of the most efficient formed in the presence of reducing agents to prevent enzyme biological systems for the detoxification of electrophilic al- inactivation. These data suggest a correlation between the redox state of the Pi class GST and its activity; this was kylating agents (3). They may also be implicated in other cellular metabolisms such as binding of hydrophobic com- recently suggested by Shaffer et al. (8)who observed that the pounds, i.e. drugs and bilirubin, steroid isomerization, and bovine placenta GST exists in an active reduced form and a less active oxidized form. These forms behave differently in reduction of hydroperoxides (3). sedimentation analysis, gel chromatography, and gel electroThe mammalian enzymes have been grouped into three distinct classes named Alpha, Mu, and Pi on the basis of phoresis (8). Unfortunately,they did not perform further several criteria including amino acid sequence, substrate, and characterizations of the oxidized form. Therefore, up to now, the term “oxidized GST” is ambiguous since the number of * The costs of publication of this article were defrayed in part by involved sulfhydryls and their identification in the primary the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 structure are notyet defined, the homogeneity of the oxidized form(s) as well as the factors affecting this process are unsolely to indicate this fact. V This paper is dedicated to Prof. Doriano Cavallini oh the occasion known. The aim of the present paper is a quantitative apof his 75th birthday proach for the characterization of the redox states of the To whom correspondence should be addressed Istituto di Scienze human placenta GST. Biochimiche, Universita “G. D’Annunzio,” Via dei Vestini 6, Chieti, We present evidence that one intrachain disulfide bond Italy. The abbreviations used GST,Glutathionetransferase; DTT, may be formed in each subunit of this transferase between DTNB, 5,5’-di- cysteines 47 and 101. This occurs in metal-catalyzed and pHdithiothreitol; CDNB, l-chloro-2,4-dinitrobenzene; thiobis-(2-nitrobenzoic acid); TNB-, thionitrobenzoate; DTDP, 4,4’- dependent processes. This oxidative reaction yields one single dithiodipyridine; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide inactive enzyme population which appears homogeneous ungel electrophoresis; Et-SH, mercaptoethanol; FPLC, fast protein liquid chromatography; HPLC, high pressure liquid chromatography; der several chromatographic and electrophoretic analyses. ANM, N-(4-anilino-l-naphthyl)maleimide; TPCK, L-l-tosyl-amido- Moreover, these techniques together with UV, circular di2-phenylethyl chloromethyl ketone. chroism, and fluorescence data let us hypothesize remarkable

21409

Redox Forms of GST

21410

conformational differences between the reduced and oxidized forms. EXPERIMENTAL PROCEDURES

Enzyme Preparation-Human placenta GST T was prepared as previously described (11).About 20 mg of this enzyme were further purified by CM52 column (2 X 10 cm) equilibrated with 10 mM Kphosphate buffer, pH 6.8 containing 1 mM EDTA. The acidic isoenzyme was not retained whereas several protein contaminants were bound to the resin. Aliquots of GST were then treated with 10 mM dithiothreitol (DTT) atpH 8.0 (0.1 M K-phosphate buffer and 1 mM EDTA) for 60 min a t 37 “C toachieve the maximal specific activity. DTT was removed by a G-25 Sephadex column (1 X 40 cm) equilibrated with 0.1 M K-phosphate buffer, pH 7.0 (orother buffers depending by the subsequent experimental conditions) and 1 mM EDTA. This activity value remains unchanged for a t least 3 h a t 37 “C (at pH 7.0) after the Sephadex chromatography. The active isoenzyme appears homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophyoresis (SDS-PAGE), disc-gel electrophoresis under nondenaturating conditions and onfastprotein liquid chromatography (FPLC). This GSTis named in this paper as “fully reduced GST” (220 units/mg). By incubating such enzyme in 0.1 M K-phosphate buffer, pH 8.0, for 48 h at 25 “C,we obtained an inactive form (1 >>1 0.018 0.053

teine residues/subunit. These data arise from the complete primary structure as deduced from the correspondent cDNA and the gene structure (20, 21). Direct titration of sulfhydryl groups in the placenta GST can be performed with thiolspecific reagents such as DTNB or DTDP although it has been observed that theclass Pi isoenzymes possess a number of masked -SH groups that do not react with these reagents even under denaturating conditions (7). In our experimental conditions the fully active enzyme has two fast reacting sulfhydryls/subunit both titrable with DTNB and DTDP and a third slow reactive thiol group (Fig. 2, Table 111).The fourth cysteine residue is not titrable in 8 M urea, 6 M guanidine, or after NaBH4 treatment and urea denaturation (Table 111).On the contrary, the fully inactivated GST (both spontaneously and copper catalyzed) has only one slow reactive -SH group/ subunit (Fig. 2, Table 111).From these data it appears that the fully inactive GST differs from the active form of two disulfides/dimer, but do not clarify whether these are intrachain or interchain bridges. On the otherhand thesedisulfides seem to involve only the four faster reacting sulfhydryls. Other interesting data arise from the -SH group titration with DTNB performed on the native enzyme at low pH values. As shown in Fig. 2 at pH 7.0 and 5.0 only four -SH groups are detectable. At pH 5.0 two fast and two slow reacting

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solvation of this transferase and occurring at low enzyme concentration (19) is negligible as appears from the experiment performed at pH7.0. The inactivated enzyme at pH9.0 after 8 h of incubation and that obtainedafter 24 h of incubation at pH8.0 have approximately the same activity of less of 1%. The time course of this process is affected by the temperature(Table I) and does not occur under nitrogen (data not shown). The presence of metal-chelating agents such as EDTA dramatically lowers the inactivation rate (Fig. 1,Table I) thereby indicating a probable involvement of metal ionsascatalysts. As shown in Fig. 1 the presence of a stoichiometric amount of cupric ions enhances about 10-fold the inactivation rate of GST. Catalytic amountsof cupric ions also yield a higher rate of inactivation, butin that case a more complex kinetic behavior has been observed. Both the spontaneous and copper-catalyzed inactivatedGSTs recovered completely the original activity by treatment with 4 mM DTT within 10 min of incubation a t pH 8.0 (37 “C). Thereactivation process is pH dependent, and it follows a pseudo-first order kinetic (Table 11).GSH yields much slower reactivation rates than DTT at all pHs tested; this could reflect both a difference in the redox potential values of the oxidized GST and glutathione or a steric hindrancefor GSH in itsreaction with the oxidized protein groups. All these data point out the probable involvement of a number of protein cysteine residues in a reversible redox process as also hypothesized for the bovine placenta GST (8) which undergoes a similar reversible inactivation in the presence of H202. Thiol Group Titration-Human placenta GST has 4 cys-

21411

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./.A

60

120

min

FIG. 2. Thiol group titration. Experimental conditions are as reported under “Experimental Procedures.” Fully reduced GST (5 p ~ reacted ) with 0.1 mM DTNB a t pH 8.0 (0);0.15 mM DTDP at pH 7.0 (A); 0.1 mM DTNB at pH 7.0 (0);0.1 mM DTNB at pH 5.0 (dotted line). Fully oxidized GST (4 p M ) reacted with 0.1 mM DTNB at pH 8.0 (B); 0.15 mM DTDP at pH 7.0 (A).In the ordinate are reported the number of titrated sulfhydryls/mol of GST (46 kDa). TABLE I11 -SH group titration data Titration procedures were performed as described under “Experimental Procedures.” pH Titrated -SH Reagent GST groups/dimer DTDP Active form 7.0 4.0 fast +slow 1.9 Active form + DTDP 7.0 6.2 fast 8 M urea Active form + DTDP 7.0 6.1 fast 8 M urea + NaBH4 DTDP 7.0 1.8 slow Inactive form DTNB Active form 8.04.2 fast + 2.0slow DTNB Active form 4.1 7.0 fast DTNB Active form 5.0 2.2 fast + 1.9slow DTNB Active form + 6.0 fast 8.0 8 M urea Active form + DTNB 8.0 6.2 fast 8 M urea + NaBH4 DTNB slowform 2.0 8.0 Inactive ~~~

Redox Forms of GST

21412

sulfhydryls can be observed.A more detailed kinetic analysis performed accordingly to Frost and Pearson (22) (data not shown) allows the calculation of apparent pseudo-first order constants of0.34 and 0.031 rnin”, respectively. During the experiment performed at pH 5.0, we also evaluated the loss of activity asa function of thetitrated sulfhydryls. The enzymatic activity was reduced to 10% when two sulfhydryls are reacted with DTNB thereby confirming the importance of only 1 reactive cysteine residue/subunit as previously observed with other thiol reagents (6, 7). The observed spontaneous disulfide formation in GST let us explore the possibility that this may also be formed during the reaction with DTNB, for example, by a thiol-disulfide exchange between a protein -SH group and theprotein-DTNB mixed disulfide.When the DTNB-reacted enzyme at pH 7.0 (four titrated -SH groups) was purified by a G-25 chromatography and treated with 1 mM DTT, the retrotitration data reported in Fig. 3 clearly indicate that four TNB- are bound to theenzyme, and therefore no protein disulfide bridgehas been formed. Electrophoresis under Denuturating and Nondenuturating Conditions-SDS-PAGE was performed both on the spontaneous and copper-inactivated GSTs and compared with the fully active enzyme. As reported under “Experimental Procedures,’’ the standardized procedure by Laemmli (16) was modified by omitting Et-SH in the sample and in the run buffer. Underthese conditionsthe active enzyme givesa single band with an apparent molecular mass of 23 kDa identical to that found in the standardized conditions with Et-SH (11) (Fig. 4).On the other hand both the inactivated forms appear as homogeneous components with the smaller apparent molecular mass of20.5 kDa. Pretreatment of the inactivated GST with 1mM DTT prior electrophoresis restores the single band at 23 kDa.The lack of bands with molecular mass higher than 23 kDa points out that no intersubunit disulfide exists in these inactivated forms. Moreover, these results suggest that the spontaneous and copper-catalyzed oxidations lead to the formation of similar homogeneous populations of inactive transferase with subunits of smaller apparent molecular mass; this may be dueto a conformational change of the molecular shape as also hypothesized for the oxidized formsof the bovine

min

FIG. 3. Retrotitration of the DTNB-treated GST. Fully re) reacted with DTNB (1mM) in 2 ml (final duced GST (1.5 p ~ were volume) of 0.1 M K-phosphate buffer, pH 7.0. The absorbance a t 412 nm reached a plateau in about 40 min corresponding to four -SH groups titrated/mol of GST. The enzyme was almost inactivated. The excess of DTNB was removed by a Sephadex G-25 column (1 X 40 cm) equilibrated with 0.1 M K-phosphate buffer, pH 8.0. The enzyme was concentrated to 2.5 ml and reacted with 1mM DTT. Theincrease of absorbance a t 412 nm was followed in continuous and at fixed times 10-pl aliquots were tested for GST activity. 0,TNB- released/ mol of GST; 0,percentage of GST activity.

I #

I /

4

1

FIG. 4. Electrophoresis under denaturating and nondenaturating conditions. Electrophoretic conditions areas reported under “Experimental Procedures.” SDS-PAGE lines a and f, molecular mass markers; Lines b and 1, fully reduced GST, lines c, d, and e, fully oxidized GST obtained by incubation a t pH 8.0, 9.0, and 8.0 stoichiometric CuS04, respectively. Lines g-i, samples of lines c-e treated with 50 mM DTT for 20 min. Disc gel electrophoresis: line 1; fully reduced GST; lines 2-4, fully oxidized GST as in lines c-e, respectively.

+

isoenzyme (8).We also observed a different chromatic intensities when identical amounts of oxidized and reduced GSTs were stained with Coomassie Brillant BlueR-250ongel. These differences werealso detected in solution by following the Bradford procedure (23); the oxidized form gives an absorption at 595 nm about 20% lower than that of the active form. This is a further indication of structural differences between these forms. The homogeneity of the GST-oxidized forms and their likeness were also supported by the disc gel electrophoresis under nondenaturating conditions (Fig. 4). Additionally in this case, the oxidized forms migrate as a single band with a higher mobility than the reduced GST. Under these conditions, however, we cannot establish whether the higher mobilityof the inactive GSTs is due to a structural modification or to anenhancement of the netnegative charge on the protein. Moreover, both the oxidized GSTs are indistinguishable on chromatofocusing analysis giving a single peak witha retention time (24 min) different from that of the active enzyme (30 min). This result is again an indication that both the spontaneously and copper-inactivatedGSTs are identical and that thedisulfide formation causes a change of the chemicophysical properties of this transferase. Identifitation of the 2 Cysteinyls Involved in the Disulfide Bridge-The spontaneously oxidized transferase was denatured in 6 M guanidinium chloride and then reacted with monoiodoacetate to alkylate other sulfhydrylsnot involved in the disulfide bond as described under “Experimental Procedures.” The modified enzyme wasthen digested withTPCKtrypsin for 24 h. After this procedure the peptide mixture was reduced with 1 mM DTT and reacted with 50 mM ANM, a specific fluorescent probe for -SH-containing compounds, recently used for the detection of the high reactive cysteine 47 of this isoenzyme (6). The HPLC pattern of this peptide mixture revealed only two majorfluorescentpeaks eluted after 29 and 33 min, both exhibiting approximately the same fluorescent intensity (Fig. 5). HPLC of the peptide mixture reacted with ANM without any DTT treatment lacks of these major fluorescent peaks, thereby indicating the absence of any free sulfhydryls in the peptides. The automated Edman degradation gave the following amino acid sequence for the first faster fluorescent peak: Asp-Gln-Gln-Glu-Ala-Ala-Leu-

Val-Asp-Met-Val-Asn-Asp-Gly-Val-Glu-Asp-Leu-Arg-XxxLys; the same analysis performed on the second fluorescent

Redox Forms of GST

21413

I

0

TIMElmnl

FIG. 6. Near U V circular dicroism spectra. Fullyoxidized GST (25 PM) was reactivated with 100 PM DTT in 2.5 ml of 0.01 M K-phosphate buffer, pH8.0 (20 "C). At fixed times 10-pl aliquots were assayed for GST activity.A, CD spectrum of the fully oxidized form plus DTT (t")(solid line) and CD spectrum of the fully reduced form (210 of min incubation, 220 units/mg) (dotted line). E, differential CD spectrum between oxidized and reduced forms. Inset, kinetics of CD perturbations at 270 nm (El) and 283 nm (0)compared with the reactivation kinetic (0);CD data are reported as percentage of maximal CD perturbations (210 min of reactivation).

FIG.5. HPLC pattern of the ANM-labeled TPCK-trypsin digest. Before HPLC analysis the fully oxidized GST wastreated as reported under "Experimental Procedures." HPLC was carried out by using a reverse-phase Aquapore RP-300 column (Applied Biosystem) (250 X 4.6-mm inner diameter). Gradientsystem was composed of 0.2% trifluoroacetic acid (solventA) and 70% acetonitrile (solvent protein disulfides are broad with no clear-cut fine structure B). Flow rate was 1 ml/min. Peptides were detected by monitoring (31). It is interesting to note that the peak centered at 270 the absorbance at 220 nm (A) and the fluorescence at 448 nm (LX = nm resembles that caused by the optically active disulfides of 355 nm) ( E ) . the oxidized forms of human somatotropin (24) and human apolipoprotein B (32). Tentatively we may assign the major peak gave the following sequence; Ala-Ser-Xxx-Leu-Tyr-Gly-band centered at 270 nm to thedisulfide of the oxidized GST Gln-Leu-Pro-Lys. By comparing these sequences with the and thesecond band at 283 nm to aromatic residues involved complete primary structure of this isoenzyme (20, 21) the 2 in some structural change. On the contrary, the far UV-CD modified cysteines can be identified as the101 and 47 residues, spectra of the oxidized and reduced forms of GST are very respectively. similar (data notshown) thereby indicating that no consistent CD Spectra-All the spectroscopic analyses were performed differences in the secondary structure occur between these on the spontaneously inactivated GST toavoid any interfer- forms. ence due to cupric ions. Moreover, since the spontaneous UV Spectra-For UV differential spectra between the reoxidation occurs at a very low rate, we always obtained duced and oxidized forms of GST, several parameters have spectroscopic data by starting with the fully oxidized GST been optimized to obtain reliable spectrophotometric data. and by following the reactivation process by DTT or Et-SH. We followed the reduction process of oxidizedGST at pH8.0 In particular, circular dichroism experiments have been per- and in the presence of 1 mM Et-SH; under these conditions formed with DTT as reducing agent since neither the reduced Et-SH which has a pKsH = 9.6 (33) is largely undissociated. DTT nor the disulfide bond in the oxidized DTT give signif- The small amount of the ionized form (about 25 p ~ is) icant dichroism over the concentrations and thespectral range stoichiometric in respect to the oxidized protein (28 p ~ ) , studied (24). From Fig. 6A, it is evident that CD spectrum of however, the presence of an high amount of undissociated Etthe fully active enzyme markedly differs from that of the SH as "buffer" makes constantthe concentration of the oxidized form in the 250-300-nm region. The interpretation dissociated form during the reduction process. These condiof such spectral changes cannot be made without an accurate tions are importantsince the thiolate ion absorbs at 230-240 knowledge of the contributions of tryptophanyls, tyrosyls, nm and any change of its concentration yields interferences and other chromophores to the totalspectrum of the reduced in the UV region even at higher wavelengths. Similarly the form of placenta GST. These data are unknown up to now use of DTT as reducing agent was avoided since its oxidized although the CD spectra of the Pi isoenzymes have been form absorbs at 283 nm. We also tested that thespontaneous reported and compared with those of the Mu and Alpha classes autoxidation of Et-SH, which leads to the formation of a isoenzymes (25). In spite of this lack several considerations disulfide absorbing at 250 nm, was negligible until the reaccan be made. First, the simple subtraction of the spectrum of tivation went to completion. As shown in Fig. 7, the reduction the reactivated GST from that of the oxidized form generates of GST is accompanied by UV perturbations in the 270-310a broad negative band with a maximum at 270 nm and a nm region. It is well known that the 270-310-nm perturbasecond smaller peak at about 283 nm (Fig. 6B),thereby tions are mainly due to changes in the environment of trypindicating at least two groups of chromophores to be involved tophyl and tyrosyl residues (34). The reduced form of GST in this spectral change. A second observation is that change shows an increased absorption in this region with maxima of the ellipticity values at 270 nm parallels the reactivation centered a t 291, 287, 285, 280, and 274 nm. A maximum kinetic (Fig. 6B, inset). It has been claimed that low energy perturbation with = 710 M" cm" was obtained after disulfide bond transitions may occur at any wavelength be- 90 min of incubation. Moreover, a broad peak can be observed tween 250-340 nm, depending on the environment of the near 300 nm. On the basis of the reported absorption differdisulfide and on the dihedral angle and thatthey can generate ence for tyrosyls and tryptophanyls due to several perturbants a conspicuous negative contribution to the ellipticity band (34), the peaks at 291, 285, and 274 nm may be due to an (26-30). Except for a few proteins, generally the CD peaks of enhanced hydrophobicity near one or both tryptophanyls of

Redox Forms of GST

21414 Ii\

1

I

toward the blue with respect to the oxidized form (Fig. 8). These differences are mainly due to tryptophanyl(s) since proportionally identical perturbations have been observed by exciting at 295 nm (data notshown). A kinetic study of these fluorescence spectra changes (Fig. 8, inset) reveals at least two distinct phenomena to occur during the reduction process. The faster blue shift of the fluorescence emission which may correspond to a movement of the tryptophanyl(s) toward a perturbation more hydrophobic environment (36); this reaches a maximum in about 60 min of incubation with 1 mM I I 270 290 310 Et-SH andparallels the reactivation kinetic. A slower quenchnm ing may bedue to a charge approach nearthe tryptophanyl(s) FIG. 7. Differential UV spectra. Fully oxidized GST (28 p ~ ) (37) and it reaches to a maximum in about 100 min reactivawereincubated with 1 mM Et-SH in 2.5 ml of 0.1 M K-phosphate tion.

buffer, pH 8.0 (25 “ C ) .BK cuvette contained all reagents except EtSH. At fixed times, 10-pl aliquots were assayed for GST activity. Dotted line, differential UV spectrum at zero time; solid line, differential UV spectrum after 90 min of reactivation. Inset, kinetics of the UV perturbations at 291 nm (0)and 300 nm (A) compared with the reactivation kinetic (0).UV data are reported as percentage of the maximal UV perturbations (100 min of reactivation). 1oc

A

3 W

0

:: 5c W

B

5 Y

1

310

,

330

,

.

350

1

,

370

1

1

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390

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FIG. 8. Fluorescence analysis. Fully oxidized GST (28 p ~were ) incubated with 1 mM Et-SH in 2.5 ml of 0.1 M K-phosphate buffer, pH 8.0 (22 “ C ) .Sample was excited at 280 nm. At fixed times 10-pl aliquots were assayed for GST activity. A, fluorescence emission spectra (LX = 280 nm) at zero time ( a ) , after 40 min of incubation ( b ) ,and after 100 min of incubation (c). B, kinetics of the fluorescence quencing (0)and blue shift (0) compared with the reactivation kinetic (0).Data are expressed as percentage of the maximal perturbations (110 min of reactivation).

the GST subunit, and the peaks at 280 and 285 nm to a similar change of polarity involving a number of tyrosyls. It is interesting to note that while these major absorption peaks increased paralleling the reactivation process, the minor peak near 300 nm follows a different kinetic pattern (Fig. 7, inset). The presence of an atypical extremum near 300 nm in solventperturbated or denaturated proteins has been explained as a signal that thetotal net charge around a tryptophyl chromophore is made less positive or more negative (35). Our data are therefore consistent with two kinetically different movements of tryptophanyls during the reduction process: a first approach of the tryptophanyl(s) to a more hydrophobic environment which causes a shift of the major absorption bands and a slower removal from a positive charge or, alternatively, a slower approach to a negative charge. Obviously, since GST has two tryptophanyls in each subunit, these two distinct modifications may reflect changes of the environment of both these aromatics as well as of a single residue. Fluorescence Spectra-Reactivation of the oxidized form of GST is accompanied by a quenching of the intrinsic protein fluorescence when the enzyme is excited at 280 nm (Fig. 8). The reduced form exhibits an emission spectrum with a maximum at 336 nm, which corresponds to a4-nmshift

CONCLUDING REMARKS

Our experimental data indicate that the human placenta GST ?r may undergo an oxidative inactivation due to an intrachain disulfide formation. This process occurs at appreciable rate only in alkaline conditions and probably requires traces of metal ions as demonstrated by the inhibiting effect of EDTA (Fig. 1).This is also confirmed by the enhancement of the oxidation rate produced by catalytic amounts of cupric ions. Both the “spontaneous” and thecopper-catalyzed processes lead to the formation of an inactive transferase which is homogeneous on SDS-PAGE, FPLC,and PAGE under nondenaturating conditions (Fig. 4). The analytical data on SDS-PAGE, performed without reducing agents, clearly indicate that no interchain disulfide bonds have been formed. Moreover, the subunit of the oxidized form of GST shows an apparent molecular mass of 20.5 kDa which is smaller than the corresponding reduced form. Since a complete recovery of the original band of 23 kDa has been observed after DTT treatment (Fig. 4)) a conformational modification maybe supposed due to a disulfide formation. This was then confirmed by the spectroscopic data whichwillbe discussed below. Our data are indicative that the oxidation involves 2 cysteines among the 4 present in each subunit. These data arise from titration experiments performed with DTNB and DTDP (Table I11 and Fig. 2). In particular, with both these reagents it appears that among the threetitrable sulfhydryls/ monomer, the 2 faster reacting cysteines are probably involved in the disulfide formation. The occurrence of a disulfide was confirmed by tryptic digestion of the oxidized transferase after blockage of the remaining free sulfhydryls with monoiodoacetate. When the peptides were reduced by DTT and labeled with ANM, only two major fluorescent peaks were detected on HPLC (Fig. 5). This is an indication that only two sulfhydryls have been oxidized in each subunit and that the disulfide is identical in both subunits. The AA sequence of these peptides allowed the identification of the cysteine 47 and the cysteine 101 as involved in this disulfide. This may be informative about the tridimensional structure of the subunit of this isoenzyme: these cysteinyls being far in the primary sequence, it seems to be reasonable that in the coiled native structure they must be sufficiently close to interact and then to form the disulfide bridge. The redox process is accompanied by changes of the tertiarystructure of this protein as it appears by several spectroscopic evidences obtained during the reduction of the fully oxidized GST. Forexample, the differential CD spectrum between the reduced and oxidized forms (Fig. 6) shows a spectral modification centered at 283 nm probably caused by a structural change near some aromatic group. The second perturbation at about 270 nm has been tentatively explained as the contribution of the disulfide to the total spectrum of

Redox Forms of GST the oxidized form. No differences have been observed in the far UV CD spectrum therebyindicating that no change in the secondary structure occurs. The perturbation of aromatic groups in the redox process of this transferase was confirmed by UV and fluorometric analyses. During the disulfide breakdown both the UV differential spectra in the 270-310 nm region (Fig. 7) and theblue shift of the intrinsic fluorescence (Fig. 8) areconsistent with a movement of one or both tryptophanyls into a more hydrophobic environment. These experiments also indicate that a second kinetically different process occurs, probably due to theapproaching of a negative charge near the tryptophan(s). All these spectroscopic data allow hypothesis of a role for the crucial cysteine 47. It has been demonstrated that the modification of this residue is incompatible with the catalytic activity of this transferase (5,6). As recently observed for the isoenzyme K from horse erythrocyte, the possibility that the inactivation is due to conformational modification by the steric hindrance of the -SH reagents is improbable as complete inactivation has been observed with many reagents of different sizes and charges (7). The involvement of this group in thecatalytic mechanism of the GST Pi cannot be excluded, but there is no evidence for that at present. On the other hand the hypothesis for a structural role of this sulfhydryl is in agreement with the data in this paper: during the redox process involving the critical cysteine 47 both inactivation and structural change have been observed simultaneously. Therefore it should be important for the maintenance of a proper tertiary structure. From another point of view these data put forth an interesting question about the possible regulation of the activity of this transferase in uiuo, based on a redox process. It has been well known that many enzymatic activities maybe modulated by redox reactions such as SH/SS exchange with biological disulfides (38-41). This has also been recently suggested for this isoenzyme (10). The present data open the new possibility that the activity of this isoenzyme may also be regulated by an intramolecular disulfide. We are now studying the possible existence of this inactive oxidized GST in several tissues and the possibility that this form is more easily attacked by proteolytic enzymes. REFERENCES 1. Chasseaud, L. F. (1979) Adu. Cancer Res. 20, 175-293 2. Jakoby, W. B., and Habig, W. H. (1980) in Enzymutic Basis of Detoxification (Jakoby, W. B., ed) Vol. 2, pp. 63-94, Academic Press, Orlando, FL 3. Mannervik, B., and Danielson, M. U. (1988) Crit. Reu. Biochem. 23,283-337 4. Mannervik, B., Alin, P., Guthenberg, C., Jensson, H., Thair, M. K., Warholm, M., and Jornvall, H. (1985) Proc. Natl. Acad. Sci. U.S. A. 8 2 , 7202-7206 5. Tamai, K., Satoh, K., Tsuchida, S., Hatayama, I., Maki, T., and Sato, K. (1990) Biochem. Biophys. Res. Commun. 1 6 7 , 331338

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