A direct spectrophotometric γ-glutamyltransferase inhibitor screening assay targeting the hydrolysis-only mode

Biochemical and Biophysical Research Communications 380 (2009) 591–596

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A direct spectrophotometric c-glutamyltransferase inhibitor screening assay targeting the hydrolysis-only mode Bjorn Vergauwen a,*, Lech W. Dudycz b, Ann Dansercoer a, Bart Devreese a a b

Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, 9000 Ghent, Belgium INFICHEM, LEX Company, 70 Bridge Street, Groton, CT 06340, USA

a r t i c l e

i n f o

Article history: Received 19 January 2009 Available online 27 January 2009

Keywords: c-Glutamyltransferase c-Glutamyl transpeptidase Glutathione High-throughput inhibitor screening Ellman’s reagent Enzyme kinetics Continuous assay a-Phenylthio-peptide derivative Thiophenol

a b s t r a c t c-Glutamyltransferase (GGT, E.C. 2.3.2.2) catalyzes the hydrolysis and transpeptidation of extracellular glutathione. Due to its central role in maintaining mammalian glutathione homeostasis, GGT is now believed to be a valuable drug target for a variety of life-threatening diseases, such as cancer. Unfortunately, however, effective tools for screening GGT inhibitors are still lacking. We report here the synthesis and evaluation of an a-phenylthio-containing glutathione peptide mimic that eliminates thiophenol upon GGT-catalyzed hydrolysis of the c-glutamyl peptide bond. The concurrent, real-time spectrophotometric quantification of the released thiophenol using Ellman’s reagent creates a GGT assay format that is simple, robust, and highly sensitive. The versatility of the assay has been demonstrated by its application to the kinetic characterization of equine kidney GGT, and enzyme inhibition assays. The ability of the glutathione mimic to behave as an excellent donor substrate (exhibiting Michaelis–Menten kinetics with a Km of 11.3 ± 0.5 lM and a kcat of 90.1 ± 0.8 nmol mg1 min1), coupled to the assay’s ability to study the hydrolysis-only mode of the GGT-catalyzed reaction, make our approach amenable to high-throughput drug screening platforms. Ó 2009 Elsevier Inc. All rights reserved.

c-Glutamyltransferase (GGT; E.C. 2.3.2.2), a cell surface heterodimeric glycoprotein, catalyses the first step in glutathione (c-L-glutamyl-L-cysteinylglycine) catabolism by hydrolyzing its cglutamyl bond. High GGT levels have been detected in kidney tubules, biliary epithelium and brain capillaries [1,2]. Also, soluble GGT can be released by such cell types in blood, where it forms GGT/lipoprotein complexes which may be of potential significance as a diagnostic/prognostic marker in cancer [3], as well as in other disease conditions such as liver dysfunction, coronary heart disease, Type 2 diabetes, stroke and atherosclerosis [4–7]. Moreover, GGT converts leukotriene C4 into leukotriene D4 [8] and has therefore been implicated in various inflammatory pathologies. During the last decade, GGT has emerged as a complex molecular discriminant in tumor cell biology (reviewed in Ref. [9]). It is generally recognized that GGT-mediated catabolism of extracellular glutathione represents an important secondary source of cysteine—and probably also of glutamate [10]—for the intracellular resynthesis of glutathione as well as for general protein synthesis [1,11]. Inside cells, glutathione plays a central role in a number of detoxification pathways for electrophilic and oxidative metabolites and xenobiotics. Hence, several tumors of GGT-positive tissues exhibit an increase in GGT expression, probably because * Corresponding author. Fax: +32 9 264 5338 E-mail address: [email protected] (B. Vergauwen). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.129

GGT-mediated cysteine-recovery confers growth and survival advantages [12,13]. While GGT clearly has been implicated in cellular antioxidant defenses [14], lines of evidence indicate that GGT plays a role in generating reactive oxygen species at the level of the cell surface as well [15,16], and, therefore, expression (also in GGTnegative cells) of GGT may play a role in human neoplasia and tumor progression [13]. In this context, GGT has emerged as an attractive target for anticancer chemotherapy, and research in this direction is currently ongoing [17,18]. The GGT-catalyzed hydrolysis of the in vivo donor substrate glutathione is outlined in Fig. 1A. GGT catalyses the transfer of the c-glutamyl moiety to an active site nucleophile to form an acyl-enzyme intermediate (acylation step of the catalytic cycle) [19,20]. This intermediate reacts subsequently with water (hydrolysis), or with an a-amino acid or dipeptide (transpeptidation), or with a new glutathione molecule (autotranspeptidation) to recover the resting GGT (deacylation step), concomitantly releasing glutamate or a c-glutamyl compound [19]. Until recently, GGT activity was studied primarily using chromogenic [21,22] or fluorigenic [23,24] substrate analogs, such as L-c-glutamyl-p-nitroanilide, from which the intensely yellow colored p-nitroaniline is liberated as a reaction product [21]. Although satisfactory for determining serum GGT levels, these kinds of assays should be avoided for enzyme mechanistic or drug screening studies, since hydrolyses, transpeptidations and autotranspeptidations occur concurrently

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Fig. 1. Overview of the proposed catalytic mechanism of GGT (A) and the reactions involved in the direct GGT assay method (B).

[19]. For example, the formation of p-nitroaniline measured as a function of substrate concentration results in non-linear (highly pH-dependent) double-reciprocal plots, due to a ‘‘substrate activation”-type mechanism, in which the donor substrate L-c-glutamyl-

p-nitroanilide accelerates the deacylation step in a concentrationdependent manner by also acting as an acceptor substrate in an autotranspeptidation reaction [25,26]. Alternatively, some methods have been described which make use of the physiological

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glutathione substrate, but these are discontinuous [27,28], require the use of radiolabeled substrates [29] and/or time consuming steps [30,31], and are therefore neither applicable in modern-day drug design projects. We report here the development of a novel highly sensitive and robust continuous colorimetric GGT assay method, highly suited for enzyme mechanistic or high-throughput drug screening studies because it uniquely allows for the study of the hydrolytic reaction in the absence of any (auto)transpeptidation reaction (hydrolysisonly mode). Materials and methods Materials and reagents S-Hexylglutathione, s-Methylglutathione, S-(4-Nitrobenzyl)glutathione, glutathione disulfide, 5,50 -dithiobis(2-nitrobenzoate) (DTNB), L-c-glutamyl-p-nitroanilide, glycylglycine, and equine kidney GGT Type II (11 U/mg solid) were purchased from Sigma–Aldrich (St. Louis, MO). Synthesis of c-glutamyl-D,L-phenylthioglycyl-glycine (H-L-Glu[DL-Gly (SPh)-Gly-OH]-OH, 8) The key chemical in the synthesis of H-L-Glu[DL-Gly(SPh)-GlyOH]-OH (8) was an acetoxy derivative as described in Ref. [32]. The complete synthesis is provided as Supplementary Material, and is outlined in the Supplementary Fig. S1. Analysis of the pH-dependent stability of c-E(PhS)GG Stock solutions of c-E(PhS)GG were prepared in water, and adjusted to a pH of 5.8. The phenylthiopeptide exhibits an absorption maximum at 258 nm at pH 7.5. The stock concentration was determined by GGT-catalyzed hydrolysis to completion and a molar extinction coefficient e258 of 2390 M1 cm1 (at pH 7.5) was calculated. The hydrolysis as a function of pH was tested using a screen of 0.1 M phosphate buffers, ranging in pH from 6.2 to 8.3. In addition, the 1-ml reaction mixtures contained 200 lM c-E(PhS)GG and 300 lM DTNB, and hydrolysis was monitored for 2 h by measuring absorbance at 412 nm using an UVIKON 933-KONTRON double beam UV/vis spectrophotometer (Secoman, France). The temperature was controlled by a Peltier thermosystem from BioTek Instruments. The pseudo first-order rate constants (kobs) for the reactions were calculated from the initial linear slopes (5–10 min time-frame). Determination of GGT activities With purified enzyme. A typical reaction with the c-E(PhS)GG donor substrate was carried out at 22 °C in a polystyrene cuvette containing 100 mM Tris–HCl (pH 7.5), 300 lM DTNB, 2–172 lM c-E(PhS)GG, 0–45 lM glutathione disulfide (GSSG) and, to initiate the reaction, 160 lg/ml equine kidney GGT. For each cE(PhS)GG-concentration, the rate of absorbance increase at 412 nm was corrected by the rate of autohydrolysis of the substrate—measured in a 3 min time-frame before the addition of GGT—to obtain the true enzymatic rate (e412 = 13600 M1 cm1), expressed as nmoles of 2-nitro-5-thiobenzoate (TNB) produced per min and per mg of GGT. This TNB producing chemical reaction is not rate limiting, as can be inferred from the linear relationship between activity and GGT concentrations up to 500 lg/ml GGT— which is more than 3-fold the concentration used in the standard reaction mixture (data not shown). The initial reaction rates were linear for at least 10 min. The L-c-glutamyl-p-nitroanilide-based

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GGT assays were essentially identical except that DTNB was omitted, and that c-E(PhS)GG was replaced by L-c-glutamyl-p-nitroanilide as the donor substrate. Activity rates were recorded at 410 nm applying an e410 of 8800 M1 cm1 [33], and were expressed as nmoles of p-nitroaniline produced per min and per mg GGT. Steady-state kinetic parameters (Km, Ki,GSSG and Vmax) were obtained by fitting the raw data to the Michaelis–Menten equation, including the influence of a competitive inhibitor, using a non-linear curvefitting program (GraphPad Prism, GraphPad, San Diego, CA). For the inhibition studies testing a number of S-substituted glutathione derivatives, the standard reaction mixture, at pH 7.5 and containing 20 lM of c-E(PhS)GG donor substrate, was supplemented with either GSSG, S-hexylglutathione, S-methylglutathione, or S(4-nitrobenzyl)glutathione, ranging in concentrations from 0 to 100 lM. Inhibition curves were fitted to the binding isotherm equation: vi = v0  (v0[I]/(IC50 + [I])), with vi, the inhibited rate, v0, the uninhibited rate, [I], the supplemented initial concentration of the S-substituted glutathione derivative, and IC50, the half maximal concentration of inhibiting substrate. The IC50 values were related to the inhibitor dissociation constants by applying the Cheng–Prusoff equation for competitive inhibition [34]. With crude samples. Sera were obtained from healthy donors and immediately used. C57BL/6 mice were a gift from Dr. G. Leclercq (Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, 9000 Ghent, Belgium). The various tissues were homogenized using a polytron homogenizer (60 s at 14,000 rpm) in 0.1 M Tris–HCl buffer, pH 8.0, and immediately used. GGT activity was assayed either with 200 lM of c-E(PhS)GG or 1 mM of L-c-glutamyl-p-nitroanilide as donor substrates, essentially as described for the purified enzyme except that 100 mM glycylglycine (pH 8.0) was included in the reaction mixtures as saturating acceptor substrate, that the Tris–HCl used had a pH of 8.0, and that the temperature was set at 37 °C. Data are expressed as mean values plusminus standard deviation from two independent experiments. The protein concentration was determined by the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA).

Results and discussion Assay design The present GGT assay was conceived through the synthesis of a novel a-phenylthio-containing glutathione peptide mimic, in which the cysteine residue is replaced by a phenylthioglycine. The synthesis of this compound, c-glutamyl-D,L-phenylthioglycylglycine (H-L-Glu[DL-Gly(SPh)-Gly-OH]-OH, or, abbreviated here, cE(PhS)GG) is outlined in Fig. S1. The assay principle is outlined in Fig. 1B; c-E(PhS)GG) is recognized by GGT as a donor substrate, releasing L-glutamate and a-phenylthioglycyl-glycine in the absence of acceptor substrates (only hydrolysis; see below) or c-glutamyl compounds and a-phenylthioglycyl-glycine in the presence of acceptor substrates (hydrolysis and transpeptidation). In any case, the spontaneous decomposition of the released a-phenylthioglycyl-glycine generates, among other compounds, thiophenol (pKa of 8 [35]), which is continuously monitored spectrophotometrically using DTNB. Since our main goal was the development of a direct GGT assay that allowed high-throughput screening for GGT inhibitors, we sought an assay method that focused on the hydrolysis reaction. In case c-E(PhS)GG would be recognized by GGT as well as reduced or oxidized glutathione (Km of 13 lM [28,29]), then mid-micromolar concentrations should be sufficient to saturate the GGT catalyzed hydrolysis reaction. Under these conditions, c-E(PhS)GG or its amino group-containing decomposition product L-Glu will hardly function as acceptor substrates in aminolysis mediated deacylation steps, since both tripeptides and amino acids

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display millimolar affinities for the acceptor site [19,29,36,37]. Hence, as has been shown for the native glutathione substrate, c-E(PhS)GG breakdown will be catalyzed by the hydrolysis-only mode, provided that no other acceptor substrates, such as glycylglycine, are included in the assay reaction mixture. Because it is well-recognized that a-heteroatom-alkyl or -arylsubstituted peptides are base-labile [38,39], the pH-dependent stability of c-E(PhS)GG was first studied spectrophotometrically in the presence of 300 lM DTNB. Rate constants for hydrolysis, kobs, recorded at either 22 or 37 °C, as a function of pH are plotted in Fig. S2A. No signs of decomposition were detected below pH 6.0 for either temperature. At 22 °C, the peptide undergoes hydrolysis with a t1/2 of 55.6 and 10.1 h at pH 7.0 and 8.0, respectively. At 37 °C, these half-lifes were 13.9 and 1.6 h, respectively. Two-hour time-courses for the hydrolysis, at pH 7.35 and 37 °C, of varying concentrations of c-E(PhS)GG are depicted in Fig. S2B, and were modeled using the first-order rate equation. The calculated kobsvalues fall within a standard deviation of 5%, thereby underscoring the system’s stability and robustness. DTNB is one of the most widely used reagents to detect and modify thiol groups of proteins. While Escherichia coli GGT does not contain a single cysteine residue, most mammalian GGT enzymes contain six cysteines, which, however, are likely not involved in catalysis [40]. To ensure that DTNB does not influence the GGT reaction, we assayed our equine kidney GGT test enzyme preparation using the standard GGT assay, during which we monitored the hydrolysis of 20 lM of the L-c-glutamyl-p-nitroanilide donor substrate, in the presence of 0, 0.3, and 0.5 mM of DTNB, with or without DTNB preincubation (37 °C, pH 7.0). All timecourses were virtually identical over a period of 30 min (data not shown), confirming an earlier report that, in this case rat, kidney GGT is DTNB insensitive [19]. Application of the assay method The non-natural tripeptidic c-E(PhS)GG was found to be an excellent substrate for our equine kidney GGT test enzyme preparation. Fig. 2 shows a detailed kinetic analysis of the GGT-catalyzed hydrolyses of c-E(PhS)GG in the absence and the presence of the cosubstrate glutathione disulfide (GSSG). For the uninhibited reaction, a Km of 11.3 ± 0.5 lM and a kcat of 90.1 ± 0.8 nmol mg1 min1 were obtained, which are kinetic constants being consistent with those for the native glutathione substrate determined earlier by radioactivity and other discontinuous assays for mammalian kidney GGT’s [28,29]. Hence, according to the rationalizations already made above, c-E(PhS)GG breakdown likely proceeds only via deacylation by hydrolysis. Furthermore, we observed a stoichiometric decomposition of our tripeptidic assay substrate, which again strongly suggests that no substrate is diverted away from hydrolytic breakdown by the aminolysis (autotranspeptidation) reaction. As expected, the cosubstrate GSSG inhibited the reaction competitively, as can be inferred from the set of straight lines that meet in a joint intercept on the y-axis upon plotting the reciprocals of the raw data (Fig. 2B). The calculated Ki of 9.55 ± 0.53 lM is in agreement with a previous report that human kidney GGT exhibited similar affinities for reduced glutathione and GSSG [40], and also corroborates with the degradation of c-E(PhS)GG exclusively being catalyzed by the hydrolysis reaction of GGT. Indeed, the Ki for GSSG inhibiting the transpeptidase reaction has been found to be 30fold higher [40]. Now knowing the true inhibition modality, it should also be possible to infer the inhibition constant for GSSG from the midpoint values of plots that are not a direct measure of affinity, such as IC50 plots, which are the approaches of choice in drug screening or structure–activity relationship campaigns. From such a dose–response plot, depicted in Fig. 2C, showing the change in the hydrolysis rate with changing concentrations of

Fig. 2. Kinetic analysis of GGT-catalyzed c-E(PhS)GG hydrolysis and determination of the inhibition modality and inhibition constant of GSSG. Initial rates of GGT activity as a function of the concentration of c-E(PhS)GG, in the absence (black squares) and in the presence of 15 (red triangles) and 45 lM (blue inverted triangles) of GSSG (A); the data are modeled using the Michaelis–Menten equation, including competitive inhibition (black continues curves). Lineweaver–Burk representation of the data from panel A (B). Initial rates of GGT activity as a function of the concentration of the inhibiting cosubstrate, GSSG (C). Initial rates of GGT activity as a function of the concentration of L-c-glutamyl-p-nitroanilide, in the absence (black squares) and in the presence of 45 lM (red diamonds) and 90 lM (blue triangles) of GSSG (D); the data were modeled using the Michaelis–Menten equation, including competitive inhibition (black continuous curves). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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GSSG, an IC50 value of 16.9 ± 1.2 lM was inferred. By applying the Cheng–Prusoff equation for competitive inhibition, we calculated a Ki of 8.65 ± 2.2 lM which is in agreement with the one determined by the detailed inhibitor/substrate titrations. Dose–response curves were also recorded for some other S-substituted glutathione derivatives for which Table 1 shows the deduced Ki-values. These inhibition constants had values around the Ki,GSSG, and, although no such kinetic parameters have been found in the literature, these results confirm earlier reports that mammalian GGT’s are highly proficient with GSSG as well as with other S-substituted glutathione derivatives [41]. To illustrate the usefulness of our assay, Fig. 2D, shows the detailed inhibitor/substrate titration study of panel A, but now using the generally used L-c-glutamyl-p-nitroanilide donor substrate. These data clearly cannot be interpreted as a reliable reflection of the inhibition modality of GSSG for the hydrolysis reaction of equine kidney GGT. Determination of GGT in human serum and in mice organs We have sought to test the applicability of our c-E(PhS)GG-based assay in the determination of GGT-catalyzed transpeptidase activity (in the presence of a saturating amount of the acceptor substrate, glycylglycine) in human serum samples and in mice organ homogenates was tested. Fig. 3 compares the values with those obtained by the conventional L-c-glutamyl-p-nitroanilide-based colorimetric Table 1 Inhibitory activities of a number of S-substituted glutathione derivatives toward equine kidney GGT, given by their respective competitive inhibition constant, Ki. S-Substituted glutathione derivative

Inhibition constant, Ki (lM)

Glutathione disulfide (GSSG) S-Methylglutathione S-Hexylglutathione S-(4-Nitrobenzyl)glutathione

8.65 ± 2.2 7.91 ± 3.2 10.3 ± 2.9 7.74 ± 1.1

Fig. 3. Application of the assay method to analyze the GGT activity in human serum (A) and tissue homogenates of mice (B).

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method. Correlation coefficients of 0.998 for the serum assays, and 0.995 for the mice organ homogenate testings demonstrate that our system is applicable to ‘‘biological” samples. In conclusion, we have developed a novel, cost-effective continuous colorimetric assay method for the determination of GGT activity. Our method features an important advantage over the conventional colorimetric or fluorigenic assays in that it does not suffer from intrinsic (auto)transpeptidation effects. The developed c-E(PhS)GG substrate is a tripeptidic glutathione mimic and is hydrolyzed by equine kidney GGT with a similar affinity and efficiency as the physiological glutathione substrate. The method therefore has the potential to facilitate and speed-up the design of highly selective GGT inhibitors. Acknowledgments This work is supported by the Research Foundation Flanders (FWO-Vlaanderen) (Grant 3G020506). B.V. is a Postdoctoral Research Fellow of the FWO-Vlaanderen. We express our gratitude to Jordi Denecker for excellent technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2009.01.129. References [1] M.H. Hanigan, W.A. Ricketts, Extracellular glutathione is a source of cysteine for cells that express gamma-glutamyl transpeptidase, Biochemistry 32 (1993) 6302–6306. [2] J.B. Whitfield, Gamma glutamyl transferase, Crit. Rev. Clin. Lab. Sci. 38 (2001) 263–355. [3] A.M. Strasak, K. Rapp, L.J. Brant, W. Hilbe, M. Gregory, W. Oberaigner, E. Ruttmann, H. Concin, G. Diem, K.P. Pfeiffer, H. Ulmer, Association of gammaglutamyltransferase and risk of cancer incidence in men: a prospective study, Cancer Res. 68 (2008) 3970–3977. [4] M. Emdin, C. Passino, A. Pompella, A. Paolicchi, Gamma-glutamyltransferase as a cardiovascular risk factor, Eur. Heart J. 27 (2006) 2145–2146. [5] D.J. Kim, J.H. Noh, N.H. Cho, B.W. Lee, Y.H. Choi, J.H. Jung, Y.K. Min, M.S. Lee, M.K. Lee, K.W. Kim, Serum gamma-glutamyltransferase within its normal concentration range is related to the presence of diabetes and cardiovascular risk factors, Diabet. Med. 22 (2005) 1134–1140. [6] I. Melezinek, J. Borovansky, M. Elleder, E. Bubnova, Tumour tissue is a source of gamma-glutamyl transpeptidase sialoform in the sera of melanoma-bearing mice, Melanoma Res. 8 (1998) 39–45. [7] A. Paolicchi, M. Emdin, C. Passino, E. Lorenzini, F. Titta, S. Marchi, G. Malvaldi, A. Pompella, Beta-lipoprotein- and LDL-associated serum gammaglutamyltransferase in patients with coronary atherosclerosis, Atherosclerosis 186 (2006) 80–85. [8] L. Orning, S. Hammarstrom, Inhibition of leukotriene C and leukotriene D biosynthesis, J. Biol. Chem. 255 (1980) 8023–8026. [9] A. Pompella, A. Corti, A. Paolicchi, C. Giommarelli, F. Zunino, Gammaglutamyltransferase, redox regulation and cancer drug resistance, Curr. Opin. Pharmacol. 7 (2007) 360–366. [10] Y.J. Kang, Y. Feng, E.L. Hatcher, Glutathione stimulates A549 cell proliferation in glutamine-deficient culture: the effect of glutamate supplementation, J. Cell. Physiol. 161 (1994) 589–596. [11] M.E. Anderson, A. Meister, Transport and direct utilization of gammaglutamylcyst(e)ine for glutathione synthesis, Proc. Natl. Acad. Sci. USA 80 (1983) 707–711. [12] S.N. Hochwald, L.E. Harrison, D.M. Rose, M. Anderson, M.E. Burt, gammaGlutamyl transpeptidase mediation of tumor glutathione utilization in vivo, J. Natl. Cancer Inst. 88 (1996) 193–197. [13] A. Pompella, V. De Tata, A. Paolicchi, F. Zunino, Expression of gammaglutamyltransferase in cancer cells and its significance in drug resistance, Biochem. Pharmacol. 71 (2006) 231–238. [14] D.R. Karp, K. Shimooku, P.E. Lipsky, Expression of gamma-glutamyl transpeptidase protects ramos B cells from oxidation-induced cell death, J. Biol. Chem. 276 (2001) 3798–3804. [15] M.J. Accaoui, M. Enoiu, M. Mergny, C. Masson, S. Dominici, M. Wellman, A. Visvikis, Gamma-glutamyltranspeptidase-dependent glutathione catabolism results in activation of NF-kB, Biochem. Biophys. Res. Commun. 276 (2000) 1062–1067. [16] S. Dominici, A. Paolicchi, A. Corti, E. Maellaro, A. Pompella, Prooxidant reactions promoted by soluble and cell-bound gamma-glutamyltransferase activity, Methods Enzymol. 401 (2005) 484–501.

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