Rat Liver Microsomal Glutathione Transferase Research Articles

Biosensing of reactive intermediates produced by the photocatalytic activities of titanium dioxide nanoparticles

The development of an enzyme based biosensing method is described for evaluating the toxicity of solutions treated by titanium dioxide photocatalysis. The method is based on the potential of rat liver microsomal glutathione transferase ability (mGST) to get enhanced in the conditions of chemical and oxidative toxicity. Phenol is taken as a model pollutant due to its toxicity and prevalence in industrial processes. Chemical analysis of the parent compound, products and acute toxicity assays using the mGST activity, were conducted during and after the various photocatalytic treatments. The maximum mGST activity was observed from 60 and 120min treated samples. This post-treatment toxicity might be due to toxic phenolic products, which may include p-benzoquinone, hydroquinone, benzenetriol and other intermediates. The enzymatic activity pattern observed after photocatalytic treatment corresponded well with the chemical degradation data obtained by HPLC-UV. The mGST assay seems to be an easy to use and promising approach for evaluating the effectiveness of wastewater treatment processes.

Activation of Rat Liver Microsomal Glutathione Transferase by Hepsin

Rat liver microsomal glutathione transferase (MGST1) is activated by limited proteolysis. Recently we purified a protease, hepsin, from rat liver microsomes that activates MGST1. In the present study the mechanism of MGST1 activation by hepsin was investigated. When MGST1 and hepsin were incubated at room temperature, MGST1 activity was markedly increased and the increase was decreased to the control level by further incubation with disulfide bond reducing agent dithiothreitol. MGST1 dimer was detected by electrophoresis after treatment of MGST1 with hepsin, instead of proteolytic product. MGST1 dimer formation accompanied by an increase in MGST1 activity was observed even in the presence of the protease inhibitor benzamidine. Furthermore, prolonged incubation of both enzymes caused the formation of MGST1 dimer and its proteolytic product. These results clearly show that the protease hepsin stimulates disulfide-linked MGST1 dimer formation resulting in activation of MGST1 and preferential degradation of MGST1 dimer. Since hepsin contains disulfide bonds in the scavenger receptor cysteine-rich (SRCR) domain, it was suggested that the SRCR domain interacts with MGST1 leading to thiol/disulfide exchange between the two enzymes followed by disulfide-linked MGST1 dimer formation.

Protection of cells from oxidative stress by microsomal glutathione transferase 1

Rat liver microsomal glutathione transferase 1 (MGST1) is a membrane-bound enzyme that displays both glutathione transferase and glutathione peroxidase activities. We hypothesized that physiologically relevant levels of MGST1 is able to protect cells from oxidative damage by lowering intracellular hydroperoxide levels. Such a role of MGST1 was studied in human MCF7 cell line transfected with rat liver mgst1 (sense cell) and with antisense mgst1 (antisense cell). Cytotoxicities of two hydroperoxides (cumene hydroperoxide (CuOOH) and hydrogen peroxide) were determined in both cell types using short-term and long-term cytotoxicity assays. MGST1 significantly protected against CuOOH and against hydrogen peroxide (although less pronounced and only in short-term tests). These results demonstrate that MGST1 can protect cells from both lipophilic and hydrophilic hydroperoxides, of which only the former is a substrate. After CuOOH exposure MGST1 significantly lowered intracellular ROS as determined by FACS analysis.

Further evidence that rat liver microsomal glutathione transferase 1 is not a cellular protein target for S-nitrosylation

By adopting biotin switch method, we recently reported that liver microsomal glutathione transferase 1 (MGST1) might not be a protein target for S-nitrosylation in rat microsomes or in vivo. However, alternative analytic methods are needed to confirm this observation, as a single biotin switch method in judging specific protein S-nitrosylation in biological samples is increasingly recognized as insufficient, or even unreliable. Besides, only MGST1 localized on endoplasmic reticulum (ER), but not mitochondria which favors protein S-nitrosylation was examined in the previous report. Present study was therefore carried out to address these issues. Primary cultured hepatocytes were used. A physiological existing nitric oxide (NO) donor S-nitrosoglutathione (GSNO) was adopted to trigger protein S-nitrosylation. MGST1 was immunoprecipitated and its S-nitrosothiol content was measured by the NO probe 2,3-diaminonaphthalene. In parallel, S-nitrosylated proteins were immunoprecipitated by a monoclonal anti- S-nitrosocysteine antibody and probed with an anti-MGST1 antibody. In hepatocytes, neither ER nor mitochondria were found to contain S-nitrosylated MGST1 after GSNO treatment, showing that differently distributed MGST1 was consistently un-nitrosylable in the cellular environment. But under broken cell conditions, when samples were incubated directly with GSNO, MGST1 S-nitrosylation was indeed detectable in both the microsomal and mitochondrial proteins, indicating that previous failure in detecting MGST1 S-nitrosylation in microsomes is due to the limitations of biotin switch method. These results clearly, if not definitely, demonstrate that MGST1 is not a ready candidate for S-nitrosylation in the cellular content, despite its susceptibility to S-nitrosylation under broken cell conditions.

Structure-activity relationship for the biotransformation of haloalkenes by rat liver microsomal glutathione transferase 1.

Many haloalkenes are nephrotoxic in rodents, and experimental evidence supports a glutathione-dependent bioactivation pathway that leads to nephrotoxicity or nephrocarcinogenicity, or both. The reaction of glutathione with haloalkenes is catalyzed by cytosolic glutathione transferases (cGST) and microsomal glutathione transferase 1 (MGST1). The aim of this study was to develop a computational approach to predict the competency of cGST and MGST1 to catalyze the reaction of glutathione with a range of haloalkenes. The hypothesis tested was that the semiempirically computed energy of the lowest unoccupied molecular orbital (E(LUMO)) of a haloalkene may be used to predict the competency of cGST and MGST1 to catalyze its reaction with glutathione. The MGST1- and cGST-catalyzed reaction of glutathione with nine haloalkenes with E(LUMO) values ranging from -1.14 to 0.38 eV was determined experimentally. The data indicated that the E(LUMO) values for haloalkenes were inversely related to the specific activity of the MGST1-catalyzed reaction but not the cGST-catalyzed reaction. These data also demonstrated that MGST1 catalyzed the reaction of glutathione with haloalkenes with E(LUMO) values equal to or more negative than -0.73 eV and that cGST catalyzed the reaction of glutathione with haloalkenes with E(LUMO) values more negative than -0.06 eV.

Binding of glutathione and an inhibitor to microsomal glutathione transferase.

Microsomal glutathione transferase is an abundant liver protein that can be activated by thiol reagents. It is not known whether the activation is associated with changed binding properties of the enzyme. Therefore the binding of GSH and an inhibitor to rat liver microsomal glutathione transferase was studied by use of equilibrium dialysis and equilibrium partition in a two-phase system. The radioactive substrate glutathione and an inhibitor (glutathione sulphonate) give hyperbolic binding isotherms with a stoichiometry of 1 mol per mol of enzyme (i.e. 1 molecule per homotrimer). Glutathione had an equilibrium binding constant of 18 microM. Competition experiments involving glutathione sulphonate showed that it could effectively displace GSH. These and kinetic studies showed that the Kd and Ki for glutathione sulphonic acid are close to 10 microM. No change in these parameters was obtained after N-ethylmaleimide activation of the enzyme. Thus activation does not result from changes in binding affinity to GSH.

Structural and Functional Aspects of Rat Microsomal Glutathione Transferase: THE ROLES OF CYSTEINE 49, ARGININE 107, LYSINE 67, HISTIDINE, AND TYROSINE RESIDUES

Rat liver microsomal glutathione transferase is rapidly inactivated upon treatment with the arginine-selective reagent phenylglyoxal or the lysine-selective 1,3,5-trinitrobenzenesulfonate. Glutathione sulfonate, an inhibitor of the enzyme, gives nearly complete protection against inactivation and prevents modification, indicating that these residues form part of or reside close to the active site. Sequence analysis of peptides from peptic and tryptic digests of [7-14C]phenylglyoxal- and 1,3,5-trinitrobenzenesulfonate-treated microsomal glutathione transferase indicated arginine 107 and lysine 67 as the sites of modification. A set of mutant forms of microsomal glutathione transferase was constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli BL21(DE3). Arginine 107 was exchanged for alanine and lysine residues. The alanine mutant (R107A) exhibited an activity and inhibition profile similar to that of the wild type enzyme but displayed a decreased thermostability. Thus, arginine 107 does not appear to participate in catalysis or substrate binding; instead, an important structural role is suggested for this residue. Lysine 67 was mutated to alanine and arginine with no effect on activity. All three histidines were replaced by glutamine, and the resulting mutant proteins had activities comparable with that of the wild type. It can thus be concluded that the chemical modification experiments indicating that arginine 107, lysine 67, and one of the histidines partake in catalysis can be disproved. However, protection from modification by a competitive inhibitor indicates that these residues could be close to the glutathione binding site. All tyrosine to phenylalanine substitutions resulted in mutants with activities similar to that of the wild type. Interestingly, the exchange of tyrosine 137 appears to result in activation of the enzyme. Thus, the microsomal glutathione transferase must display an alternate stabilization of the thiolate anion of glutathione other than through interaction with the phenolic hydroxyl group of a tyrosine residue. Substitution of cysteine 49 with alanine resulted in a semiactivated mutant enzyme with enzymatic properties partly resembling the activated form of microsomal glutathione transferase. The function of this mutant was not altered upon reaction with N-ethylmaleimide, and cysteine 49 is thus demonstrated as the site of modification that results in activation of microsomal glutathione transferase.

Membrane Topology of Recombinant Rat Liver Microsomal Glutathione Transferase Expressed inE. coli

Rat liver microsomal glutathione transferase is a mammalian membrane protein that can be successfully expressed inEscherichia coliin an enzymatically active form. The protein does not form inclusion bodies and is recovered in the membrane fraction. The membrane topology of recombinant rat liver microsomal glutathione transferase expressed inE. coliwas investigated by comparing the proteolytic cleavage products from intact and permeabilized spheroplasts. It was shown that lysine-4 of microsomal glutathione transferase is directed towards the outside, whereas lysine-41 faces the inside of theE. coliinner membrane. This shows that microsomal glutathione transferase has an inside-out orientation inE. colispheroplasts as compared to liver microsomes. This fact enables us to make topology experiments that were previously not possible. Intact spheroplasts treated with pronase yielded a cleavage pattern consistent with two additional exposed segments closer to the C-terminus. Thus a polytopic model is suggested for the membrane association of microsomal glutathione transferase.

Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli.

The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.

Ontogenesis of rat liver microsomal glutathione transferase

The ontogenesis of rat liver microsomal glutathione transferase was investigated by activity measurements and immunochemical methods. The activity rises from a very low level (3% of adults) at day 8 pre-partum to adult levels at days 50–150. Increases are associated with the neonatal and late-suckling clusters. Interestingly the capacity to become activated by N-ethylmaleimide is much lower in females early and late in life (days 35–100 and 300–550). After the initial increases (from 10% of adult levels at day 8 pre-partum), protein levels determined immunochemically remain constant throughout life with no apparent sex differences. The developmental pattern of microsomal glutathione transferase resembles those of other drug-metabolizing enzymes indicating that the function of the enzyme is required in adult life.

Microsomal glutathione transferase: Lipid-derived substrates and lipid dependence

Rat liver microsomal glutathione transferase was found to display glutathione peroxidase activity toward a variety of oxidized lipids. 1-Linoleoyl-2-palmitoyl phosphatidylcholine hydroperoxide, 2-linoleoyl-1-palmitoyl phosphatidylcholine hydroperoxide, 2-linoleoyl-1-palmitoyl phosphatidylethanolamine hydroperoxide, and cholesteryl linoleate hydroperoxide all served as substrates (0.02, 0.04, 0.02, and 0.02 μmol/min mg, respectively). The phospholipid hydroperoxide glutathione peroxidase activity of the enzyme was found not require detergent and increased when liposomes containing peroxidized phospholipid were fused with liposomes containing microsomal glutathione transferase. Methyl linoleate ozonide serves as a very efficient substrate for the microsomal glutathione transferase. The unactivated and N-ethylmaleimide-activated enzyme displayed specific activities of 0.74 and 5.9 μmol/min mg, respectively. Upon examination of a series of 4-hydroxyalk-2-enals it was found that the catalytic efficiency of the enzyme increases from the 4-hydroxyhept-2-enal up to the 4-hydroxytetradec-2-enal. The specific activities with the various 4-hydroxyalk-2-enals tested varied between 0.28 and 0.95 μmol/min mg. The phospholipid dependence of the microsomal glutathione transferase was examined in proteoliposomes formed by cholate dialysis. Phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, and rat liver microsomal phospholipids could all be used successfully to reconstitute the enzyme. In conclusion, microsomal glutathione transferase can detoxify a number of lipid peroxidation products as well as fatty acid ozonide. The results imply a protective role for the enzyme under conditions of oxidative stress.

Kinetic studies on rat liver microsomal glutathione transferase: consequences of activation

Rat liver microsomal glutathione transferase is activated by sulfhydryl reagents and proteolysis. This property varies, however, depending on the combination, concentration and reactivity of the substrates. Thus, a multi-dimensional diagram can be envisioned in which the parameters affecting enzyme activity and activation are visualized. In principle activation could stem from an alteration in enzyme mechanism, transition-state complementarity, product release rate or pH-rate behaviour. These studies appear to rule out these possibilities and an alternate hypothesis is suggested based on the following experiments: (i) alternate substrate diagnosis of the kinetic mechanism of microsomal glutathione transferase indicates a random sequential mechanism. Non-activated and activated enzyme follow the same mechanism by these criteria. (ii) The microsomal glutathione transferase stabilizes a Meisenheimer complex between 1,3,5-dinitrobenzene and glutathione. The formation constants were similar for the unactivated and activated enzyme ((15 ± 1) · 10 3 and (14 ± 1)· 10 3 M −, respectively, at pH 8). Inasmuch as the Meisenheimer complex resembles the transition state there is no evidence for an increased stabilization upon activation. (iii) The catalytic rate constant k cat, does not vary with the viscosity in the assay medium. Thus, product release is not rate limiting for the unactivated and activated microsomal glutathione transferase (with saturating 1-chloro-2,4-dinitrobenzene and varying GSH). (iv) The pH dependence of the K f-values for Meisenheimer complex formation exhibited pK a values close to 6 for both the activated and unactivated microsomal glutathione transferase. The pH profile of k cat (with saturating 1-chloro-2,4-dinitrobenzene and variable GSH concentrations) showed apparent pK a values of 5.7 ± 0.5 and 6.3 ± 0.4 for the unactivated and activated enzyme, respectively, indicative of a very similar requirement for deprotonation of the enzyme-GSH-1-chloro-2,4-dinitrobenzene complex. (v) Examination of the kinetic parameters (obtained with GSH as the variable substrate against increasingly reactive electrophilic substrates) in Hammett plots shows that the activation mechanism entails a more efficient utilization of GSH. It is suggested that a higher rate of formation of the glutathione thiolate anion occurs in the activated enzyme.

Functional and structural membrane topology of rat liver microsomal glutathione transferase

The membrane topology of rat liver microsomal glutathione transferase was investigated by comparing the tryptic cleavage products from intact and permeabilized microsomes. It was shown that lysine-4 of microsomal glutathione transferase is accessible at the luminal surface of the endoplasmic reticulum, whereas lysine-41 faces the cytosol. These positions are separated by a hydrophobic stretch of 25 amino acids (positions 11–35) which comprises the likely membrane-spanning region. Reaction of cysteine-49 of the microsomal glutathione transferase with the charged sulfhydryl reagent DTNB (2,2′-dithiobis(5-nitrobenzoic acid))) in intact microsomes further supports the cytosolic localization of this portion of the polypeptide chain. The role of two other potential membrane-spanning/associated segments in the C-terminal half of the polypeptide chain was examined by investigating the association of the protein to the membrane after trypsin cleavage at lysine-41. Activity measurements and Western blot analysis after washing with high concentrations of salt, as well as after phase separation in Triton X-114, indicate that this portion of the protein also binds to the membrane. It is also shown that cleavage of the purified protein at Lys-41 and subsequent separation of the fragments obtained yields a functional C-terminal polypeptide with the expected length for the product encompassing positions 42–154. The location of the active site of microsomal glutathione transferase was investigated using radiolabelled glutathione together with a second substrate. Since isolated rat liver microsomes do not take up glutathione or release the glutathione conjugate into the lumen, it can be concluded that the active site of rat liver microsomal glutathione transferase faces the cytosolic side of the endoplasmic reticulum.

Identification of N-acetylcysteine as a new substrate for rat liver microsomal glutathione transferase. A study of thiol ligands.

N-Acetyl-L-cysteine serves as an efficient substrate for the rat liver microsomal glutathione transferase with 1-chloro-2,4-dinitrobenzene as second substrate (8.8 +/- 0.37 mumol/min mg). The activity is actually higher than that obtained with glutathione (2-4 mumol/min mg). In examining the activity of liver subcellular fractions, no activity with N-acetyl-L-Cys could be detected in dialyzed or N-ethylmaleimide-treated (in order to remove endogenous glutathione) cytosol. The activity in rat liver microsomes was 0.11 +/- 0.007 mumol/min mg, which is accounted for by the content of microsomal glutathione transferase. Thus, N-acetyl-L-Cys can be used as a specific substrate for determining the conjugating activity of microsomal glutathione transferase. N-Acetyl-L-Cys was also shown to function as a substrate for the enzyme when other second substrates than 1-chloro-2,4-dinitrobenzene (with varying electrophilicity) are used. The pH dependence of microsomal glutathione transferase was studied. The kcat/Km(1-chloro-2,4-dinitrobenzene) was dependent on pH with an apparent pKa of 6, > or = 9, and > or = 8 with saturating glutathione, gamma-L-Glu-L-Cys, and N-acetyl-L- cysteine, respectively. Apparently the enzyme has the ability to lower the pKa of glutathione by 3 orders of magnitude. The kcat/Km(thiol) did not vary appreciably with pH (except for N-acetyl-L-cysteine), indicating that no rate-determining deprotonation occurs on the enzyme itself between pH 5.5 and 9. The abilities of histidine-, lysine-, and arginine-selective reagents to inactivate the enzyme when N-acetyl-L-cysteine and gamma-L-Glu-L-Cys were used as substrates were investigated. The activity toward N-acetyl-L-cysteine was decreased considerably less after treatment with the arginine-selective reagent phenylglyoxal, as compared to the activity toward GSH and gamma-L-Glu-L-Cys. This indicates that an arginine makes contact with gamma-L-Glu residue in GSH. With the other reagent/substrate combinations tested the enzyme was inactivated almost completely. The ability of microsomal glutathione transferase to stabilize the Meisenheimer complex formation between 1,3,5-trinitrobenzene and various glutathione analogues, including non-substrate thiols, has been examined. It is shown that, in general, substrates exhibited higher formation constants (approaching 50 mM-1) than non-substrates (4.5 +/- 1.7 mM-1, n = 7), whereas simpler thiols did not yield enzyme-bound complexes. The fact that the enzyme can stabilize Meisenheimer complexes from non-substrate thiol analogues of glutathione offers new possibilities for examining the substrate interactions of glutathione transferases.

Evidence that rat liver microsomal glutathione transferase is responsible for glutathione-dependent protection against lipid peroxidation

Evidence that rat liver musomal glutathione transferase is responsible for the glutathione-dependent inhibition of lipid peroxidation in liver musomes has been obtained. Activation of the musomal glutathione transferase in musomes by cystamine renders this organelle even more resistant to lipid peroxidation in the presence of glutathione compared with untreated musomes. Upon examining the effect of seven glutathione analogues on lipid peroxidation, it was found that only those that serve as good substrates for the musomal glutathione transferase (Glutaryl- l-Cys-Gly and α- l-Glu- l-Cys-Gly) can inhibit lipid peroxidation. The lack of inhibition by the other five analogues (α d-Glu- l-Cys-Gly, γ- d-Glu- l-Cys-Gly, β- l-Asp- l-Cys-Gly, α- l-Asp- l-Cys-Gly and α- d-Asp- l-Cys-Gly) shows the specificity of the protection and rules out any non-enzymic component. Inhibitors of selenium- dependent glutathione peroxidase (mercaptosuccinate at 50 μM) and phospholipid hydroperoxide glutathione peroxidase (iodoacetate, 1 mM + glutathione, 0.5 mM) do not inhibit the glutathione-dependent protection of rat liver musomes against lipid peroxidation. Purified musomal glutathione transferase, NADPH-cytochrome P450 reductase and cytochrome P450 were reconstituted in musomal phospholipid vesicles by cholate dialysis. The resulting membranes contained functional enzymes and did display enzymic lipid peroxidation induced by 75 μM NADPH and 10 μM Fe-EDTA (2: 1). This model system was used to investigate whether musomal glutathione transferase could inhibit lipid peroxidation in a glutathione-dependent manner. The results show that 5 mM glutathione did inhibit lipid peroxidation when functional musomal glutathione transferase was included. This was not the case when the enzyme had been pre-inactivated with diethylpyrocarbonate. Furthermore, the protective effect of glutathione could be partly reversed by an inhibitor (100μM bromosulphophtalein) of the enzyme. Apparently, rat liver musomal glutathione transferase has the capacity to inhibit lipid peroxidation in a reconstituted system.

The oligomeric structure of rat liver microsomal glutathione transferase studied by chemical cross-linking

The oligomeric structure of rat liver microsomal glutathione transferase was investigated using four different chemical cross-linking reagents. Studies were performed with the isolated enzyme, with the enzyme incorporated into phosphatidyl choline liposomes and with rat liver microsomes. Cross-linking was analyzed by use of SDS-PAGE combined with Western blotting. Our results strongly suggest that the microsomal glutathione transferase is a trimer in situ in the endoplasmic reticulum, as well as in the purified state and in proteoliposomes. These results lend strong support to previous studies, involving hydrodynamic characterization and radiation inactivation, indicating that the microsomal glutathione transferase is a trimeric enzyme.

Mechanism of activation of rat liver microsomal glutathione transferase by noradrenaline and xanthine oxidase

Activation of glutathione transferase activity in rat liver microsomes under a variety of conditions producing oxidative stress was investigated. Neither hydrogen peroxide (10mM) (added or produced endogenously by glucose + glucose oxidase) nor duroquinone together with an NADPH- regenerating system (which generates the superoxide anion radical) had any significant effect on the glutathione transferase activity towards 1-chloro-2, 4-dinitrobenzene. On the other hand, incubation of microsomes with 1mM noradrenaline (which autooxidizes and generates superoxide anion radical) gave a 160% activation, as shown earlier (Aniya and Anders, J Biol Chem 264: 1998–2002, 1989. This was taken as an indication that microsomal glutathione transferase could be activated by oxidative stress. Here, we demonstrate that activation by this compound is due to covalent binding (presumably of the quinone formed during autooxidation). The xanthine/xanthine oxidase system, which generates the superoxide anion radical and hydrogen peroxide, increases microsomal glutathione transferase activity, but this activation was not dependent on the presence of xanthine. Western blots of microsomes treated with xanthine oxidase revealed that activation was due to proteolysis (presumably by contaminating proteases in the xanthine oxidase). In conclusion, there is no firm evidence that rat liver microsomal glutathione transferase is activated directly by reduced oxygen species in the microsomal system. The possibility remains that oxidative stress triggers secondary mechanisms such as generation of reactive intermediates and/or activation of proteolysis, which can in turn increase enzyme activity.

Studies on the activation of rat liver microsomal glutathione transferase in isolated hepatocytes

The mechanism of activation of microsomal glutathione transferase in isolated liver cells by diisapropylidene acetone (phorone) was investigated. Phorone (1 mM) causes a time-dependent increase (up to 2.6-fold) in the glutathione transferase activity of microsomes isolated from treated hepatocytes. Since phorone reacts with sulfhydryl groups, the possibility that this compound activated microsomal glutathione transferase directly was studied. It was found that neither the activity of the purified enzyme nor that in isolated microsomes is affected by phorone. It has been suggested [Masukawa T and Iwata H, Biochem Pharmacol 35: 435–438, 1986] that activation of microsomal glutathione transferase by phorone in vivo is mediated through thiol-disulfide interchange involving oxidized glutathione (GSSG). It is shown here that the glutathione transferase activity of isolated microsomes, which was increased by the addition of 10 mM GSSG, can be decreased to the basal level with 0.1 M dithioerythritol. Dithioerythritol, on the other hand, only marginally decreases the glutathione transferase activity in microsomes isolated from phorone-treated hepatocytes. This finding argues against a role for thioldisulfide interchange in the activation of the enzyme by phorone. Furthermore, the glutathione depletion caused by phorone does not seem to be responsible for activation per se, since other thiol depletors [e.g. diethylmaleate (DEM)] do not affect the activity of the enzyme. Immunoblot analysis of microsomes isolated from phorone-treated hepatocytes did not reveal any partial proteolysis which might have accounted for the activation. It is suggested that activation of microsomal glutathione transferase by phorone proceeds through a mechanism which might reflect an in vivo regulation of this enzyme. Additional compounds which have been shown to activate the microsomal glutathione transferase in vivo were also tested and significant activation was obtained with 1,2-dibromoethane (1.4-fold) but not with DEM or carbon tetrachloride. Activation was also obtained with 1-chloro-2,4-dinitrobenzene (CDNB) (1.6-fold) and to a small extent with t-butyl hydroperoxide (1.2-fold). The activation by 1,2-dibromoethane and CDNB is probably mediated through covalent binding, considering the known alkylating properties of these compounds. CDNB is the first substrate shown to activate the microsomal glutathione transferase implying that electrophilic compounds which are substrates can increase the rate of their own elimination by reacting with this enzyme. In addition, activation by t-butyl hydroperoxide indicates that oxidative stress can activate microsomal glutathione transferase.

Purification of rat liver microsomal glutathione transferase

The rat liver microsomal glutathione transferase was activated by N-ethylmaleimide, solubilized by Triton X-100 and purified by chromatography on hydroxyapatite and CM Sephadex C-50. A 28-fold purification resulted in a 38% yield. The purified protein moved as a band with an apparent molecular weight of 14,000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis and appeared to be nearly homogeneous. The N-terminal amino acid of the purified enzyme was alanine, identified by the dansyl method. Optimum pH and temperature were 6.8 and 30 degrees C, respectively.

Studies on the activity and activation of rat liver microsomal glutathione transferase with a series of glutathione analogues

The substrate specificity of rat liver microsomal glutathione transferase toward glutathione has been examined in a systematic manner. Out of a glycyl-modified and eight gamma-glutamyl-modified glutathione analogues, it was found that four (glutaryl-L-Cys-Gly, alpha-L-Glu-L-Cys-Gly, alpha-D-Glu-L-Cys-Gly, and gamma-L-Glu-L-Cys-beta-Ala) function as substrates. The kinetic parameters for three of these substrates (the alpha-D-Glu-L-Cys-Gly analogue gave very low activity) were compared with those of GSH with both unactivated and the N-ethylmaleimide-activated microsomal glutathione transferase. The alpha-L-Glu-L-Cys-Gly analogue is similar to GSH in that it has a higher kcat (6.9 versus 0.6 s-1) value with the activated enzyme compared with the unactivated enzyme but displays a high Km (6 versus 11 mM) with both forms. Glutaryl-L-Cys-Gly, in contrast, exhibited a similar kcat (8.9 versus 6.7 s-1) with the N-ethylmaleimide-treated enzyme but retains a higher Km value (50 versus 15 mM). Thus, the alpha-amino group of the glutamyl residue in GSH is important for the activity of the activated microsomal glutathione transferase. These observations were quantitated by analyzing the changes in the Gibbs free energy of binding calculated from the changes in kcat/Km values, comparing the analogues to GSH and each other. It is estimated that the binding energy of the alpha-amino group of the glutamyl residue in GSH contributes 9.7 kJ/mol to catalysis by the activated enzyme, whereas the corresponding value for the unactivated enzyme is 3.2 kJ/mol. The importance of the acidic functions in glutathione is also evident as shown by the lack of activity with 4-aminobutyric acid-L-Cys-Gly and the low kcat/Km values with gamma-L-Glu-L-Cys-beta-Ala (0.03 and 0.01 mM-1s-1 for unactivated and activated enzyme, respectively). Utilization of binding energy from a correctly positioned carboxyl group in the glycine residue (10 and 17 kJ/mol for unactivated and activated enzyme, respectively) therefore also appears to be required for optimal activity and activation. A conformational change in the microsomal glutathione transferase upon treatment with N-ethylmaleimide or trypsin, which allows utilization of binding energy from the alpha-amino group of GSH as well as the glycine carboxyl in catalysis, is suggested to account for at least part of the activation of the enzyme.