Liver Microsomal Glutathione S-transferase Research Articles

Reactive nitrogen species derived activation of rat liver microsomal glutathione S-transferase

The effect of reactive nitrogen species on rat liver microsomal glutathione S-transferase (MGST1) was investigated using microsomes and purified MGST1. When microsomes or the purified enzyme were incubated with peroxynitrite (ONOO −), the GST activity was increased to 2.5–6.5 fold in concentration-dependent manner and a small amount of the MGST1 dimer was detected. MGST1 activity was increased by ONOO − in the presence of high amounts of reducing agents including glutathione (GSH) and the activities increased by ONOO − or ONOO − plus GSH treatment were decreased by 30–40% by further incubation with dithiothreitol (DTT, reducing disulfide) or by sodium arsenite (reducing sulfenic acid). Furthermore, GSH was detected by HPLC from the MGST1 which was incubated with ONOO − plus GSH or S-nitrosoglutathione followed by DTT treatment. In addition, the MGST1 activity increased by nitric oxide (NO) donors such as S-nitrosoglutathione, S-nitrosocysteine or the non-thiol NO donor 1-hydroxy-2-oxo-3 (3-aminopropyl)-3-isopropyl was restored by the DTT treatment. Since DTT can reduce S-nitrosothiol and disulfide bond to thiol, S-nitrosylation and a mixed disulfide bond formation of MGST1 were suggested. Thus, it was demonstrated that MGST1 is activated by reactive nitrogen species through a forming dimeric protein, mixed disulfide bond, nitrosylation and sulfenic acid.

Purification of Liver Serine Protease Which Activates Microsomal Glutathione S-Transferase: Possible Involvement of Hepsin

Rat liver microsomal glutathione S-transferase (MGST1) is known to be activated by trypsin, however, it has not been clarified whether MGST1 is activated by a protease present in liver. In the present study we purified the MGST1 activating protease from liver microsomes and finally identified that the protease is hepsin, a type II transmembrane serine protease. When the protease was incubated with the purified MGST1 or liposomal MGST1 at 4 degrees C, MGST1 activity was increased 3-4.5 fold after 3-6 d. In electrophoretic and immunoblot analyses after the incubation of MGST1 with the protease MGST1 dimer and its degraded fragment were detected. These results suggest that the rat liver microsomal hepsin functions as MGST1 activating/degrading enzyme.

Activation of rat liver microsomal glutathione S-transferase by gallic acid

The effect of phenolic antioxidants on the rat liver microsomal glutathione S-transferase (MGST1) was investigated in vitro. When microsomes were incubated with various polyphenolic antioxidants, gallic acid (3,4,5-trihydroxybenzoic acid) markedly increased MGST1 activity and the increase was prevented in the presence of superoxide dismutase (SOD) or catalase. The MGST1 activity increased by gallic acid was decreased by further incubation with sodium arsenite, a sulfenic acid reducing agent, but was not with dithiothreitol, a disulfide bond reducing agent. The incubation of microsomes with gallic acid in the presence of the NADPH generating system which generates reactive oxygen species (ROS) through cytochrome P-450 system increased the MGST1activity in spite of scavenging the ROS and the increase was also depressed by SOD/catalase. The increase of MGST1 activity by gallic acid was prevented by co-incubation with a stable radical, 1,1-diphenyl-2-picrylhydrazyl or ferric chloride. These results suggest that the gallic acid acts as a pro-oxidant and activates MGST1 through oxidative modification of the enzyme.

Metabolism of melphalan by rat liver microsomal glutathione S-transferase

One of the major problems in the treatment of human cancer is the phenomenon of drug resistance. Increased glutathione (γ-glutamylcysteinylglycine, GSH) conjugation (inactivation) due to elevated level of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) remains uncertain. In our experiments, a combination of liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between GSH and melphalan, one of the alkylating agents. The spontaneous reaction of 1 mM melphalan with 5 mM GSH at 37 °C in aqueous phosphate buffer for 1 h gave primarily the monoglutathionyl and diglutathionyl melphalan derivatives, with small amounts of mono- and dihydroxy melphalan derivatives. We demonstrated that rat liver microsomal GST presented a strong catalytic effect on the reaction as determined by the increase of monoglutathionyl and diglutathionyl melphalan derivatives and the decrease of melphalan. We showed that microsomal GST was activated by melphalan in a concentration- and time-dependent manner. Microsomal GST which was stimulated approximately 1.5-fold with melphalan had a stronger catalytic effect. Thus microsomal GST may play a potential role in the metabolism of melphalan in biological membranes, and in the development of ADR.

Microsomal Glutathione S-Transferase

Rat liver microsomal glutathione S-transferase was activated with N-ethylmaleimide, solubilized with Triton X-100, and purified by chromatography on hydroxyapatite and CM-Sepharose. A 36-fold purification resulted in a 36% yield, indicating that the glutathione S-transferase accounts for 2.5-3% of the original microsomal protein. The purified protein moved as a band with an apparent molecular weight of 14 000 on sodium dodecyl sulphate gel electrophoresis and appeared to be nearly homogeneous. The complex formed between the purified microsomal glutathione S-transferase and Triton X-100 has a sedimentation coefficient of 3.2 S, a partial specific volume of 0.844 cm3/g, and a Stokes radius of 5.5 nm. The complex has a molecular weight of 127 000 and contains three or four polypeptide chains and 112-134 detergent molecules. Antibodies directed against soluble glutathione S-transferases A, B and C do not react with the purified microsomal enzyme. This finding, together with differences in molecular weight and substrate specificity, demonstrate that the microsomal glutathione S-transferase is an enzyme distinct from the cytosolic glutathione S-transferases.

Metabolism of chlorambucil by rat liver microsomal glutathione S-transferase

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil (4-[ p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid; CHB), is often limited by the emergence of drug resistant tumor cells. Increased glutathione (γ-glutamylcysteinylglycine; GSH) conjugation (inactivation) of alkylating anticancer drugs due to overexpression of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance to alkylating agents. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) to CHB remains uncertain. In our experiments, a combination of lipid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between CHB and GSH. The spontaneous reaction of 1 mM CHB with 5 mM GSH at 37 °C in aqueous phosphate buffer for 1 h gave primarily the monoglutathionyl derivative, 4-[ p-[ N-2-chloroethyl, N-2- S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG) and the diglutathionyl derivative, 4-[ p-[bis[2- S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG 2) with small amounts of the hydroxy-derivative, 4-[ p-[ N-2- S-glutathionylethyl, N-2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBSGOH), 4-[ p-[bis[2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBOH 2), 4-[ p-[ N-2-chloroethyl, N-2- S-hydroxyethyl]amino]phenyl]-butanoic acid (CHBOH). We demonstrated that rat liver microsomal GST presented a strong catalytic effect on these reactions as determined by the increase of CHBSG 2, CHBSGOH and CHBSG and the decrease of CHB. We showed that microsomal GST was activated by CHB in a concentration and time dependent manner. Microsomal GST which was stimulated approximately two-fold with CHB had a stronger catalytic effect. Thus, microsomal GST may play a potential role in the metabolism of CHB in biological membranes, and in the development of ADR.

Activation of microsomal glutathione s-transferase by peroxynitrite.

Peroxynitrite (ONOO-) toxicity is associated with protein oxidation and/or tyrosine nitration, usually resulting in inhibition of enzyme activity. We examined the effect of ONOO- on the activity of purified rat liver microsomal glutathione S-transferase (GST) and found that the activity of reduced glutathione (GSH)-free enzyme was increased 4- to 5-fold by 2 mM ONOO-; only 15% of this increased activity was reversed by dithiothreitol. Exposure of the microsomal GST to ONOO- resulted in concentration-dependent oxidation of protein sulfhydryl groups, dimer and trimer formation, protein fragmentation, and tyrosine nitration. With the exception of sulfhydryl oxidation, these modifications of the enzyme correlated well with the increase in enzyme activity. Nitration or acetylation of tyrosine residues of the enzyme using tetranitromethane and N-acetylimidazole, respectively, also resulted in increased enzyme activity, providing additional evidence that modification of tyrosine residues can alter catalytic activity. Addition of ONOO--treated microsomal GST to microsomal membrane preparations caused a marked reduction in iron-induced lipid peroxidation, which raises the possibility that this enzyme may act to lessen the degree of membrane damage that would otherwise occur under pathophysiological conditions of increased ONOO- formation.

Regulation of microsomal and cytosolic glutathione S-transferase activities by S-nitrosylation

There is increasing evidence that S-nitrosylation is a mechanism for the regulation of protein function via the modification of critical sulfhydryl groups. The activity of rat liver microsomal glutathione S-transferase (GST) is increased after treatment with N-ethylmaleimide (NEM), a sulfhydryl alkylating reagent, and is also increased under conditions of oxidative stress. In the present study, preincubation of purified rat liver microsomal GST with S-nitrosoglutathione (GSNO) or the nitric oxide (NO) donor, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), resulted in a 2-fold increase in enzyme activity. This increase in activity was reversed by dithiothreitol. The initial treatment of microsomal GST with either GSNO or DEA/NO was associated with an 85% loss of free sulfhydryl groups. After removal of the nitrosylating agents over a 6-hr period, approximately 50% of the enzyme was still nitrosylated, as determined by redox chemiluminescence. Furthermore, preincubation of either purified enzyme or hepatic microsomes with GSNO or DEA/NO prevented further enzyme activation by NEM, suggesting that NEM and the NO donors interact with a common population of sulfhydryl groups in the enzyme. In contrast, both NEM and NO donors partially inhibited the activity of cytosolic GST isoforms. The inhibitory activity of NEM and NO donors was much more evident when the GST pi isoform was used instead of a mixture of GST isoforms. These data suggest that there may be differential regulation of microsomal and cytosolic GST activities under conditions of nitrosative stress.

Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization

A 25 kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na(2)CO(3), indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73-83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3 Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243-248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87-101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H(2)O(2). Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.

Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization

A 25kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na2CO3, indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73–83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243–248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87–101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H2O2. Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.

Cloning, characterisation and bacterial expression of full length cDNA for the mouse liver microsomal glutathione S-transferase.

We have isolated a cDNA encoding full length microsomal glutathione S-transferase (MGST) from mouse liver. The cDNA was isolated by RT-PCR using primers designed from published cDNA sequence of rat MGST with the addition of 5' Nde-1 and 3' HindIII sites, and cloned into bacterial expression vector pSP19T7LT. Deduced amino acid sequence (155 amino acids, calculated mol.mass 17512 Dalton) confirmed the identity of microsomal GST from mouse liver which has sequence homology with that of rat and human liver MGST1. Recombinant GST cDNA (Genbank accession # 159050) was expressed in BL21(DE3) in the presence of 1 mM IPTG at 30 degrees C. The expressed GST protein was found to be localised in the bacterial membrane as determined by measuring catalytic activity using CDNB and cumene hydroperoxide substrates, SDS-PAGE and Western blot analysis. We have demonstrated the cloning and expression of full length cDNA for MGST from mouse liver and have characterised the functionally active product as MGST protein. These results should facilitate studies on the role of MGST in the regulation of chemical carcinogenesis and in the prevention of oxidative stress caused by endogenous and exogenous chemicals.

Dimerumic acid as an antioxidant of the mold, Monascus Anka

We previously reported that the mold Monascus anka, traditionally used for fermentation of food, showed antioxidant and hepatoprotective actions against chemically induced liver injuries. In the present study, the antioxidant component of M. anka was isolated and identified. The antioxidant was elucidated to be dimerumic acid. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical was significantly scavenged by the antioxidant whereas hydroxyl radical and superoxide anion were moderately scavenged. When the antioxidant (12 mg/kg) was given to mice prior to carbon tetrachloride (CCl 4, 20 μl/kg, ip) treatment, the CCl 4-induced liver toxicity in mice seen in an elevation of serum aspartate aminotransferase and alanine aminotransferase activities was depressed, suggesting the hepatoprotective action of the antioxidant. The liver microsomal glutathione S-transferase activity, which is known to be activated by oxidative stress or active metabolites, was increased by CCl 4 treatment and the increase was also depressed by pretreatment with the mold antioxidant. Thus these data confirmed that the dimerumic acid isolated from M. anka is the potential antioxidant and protective against CCl 4-induced liver injury.

Microsomal formation of S-nitrosoglutathione from organic nitrites: possible role of membrane-bound glutathione transferase

The formation of S-nitrosoglutathione (GSNO) from amyl nitrite and n-butyl nitrite was studied in rat liver microsomes, employing N-ethylmaleimide (MalNEt) as an activator and indomethacin as an inhibitor of microsomal glutathione S-transferase (GST). Rates were compared with GST activity measured with 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. MalNEt stimulated GST activity and the formation of GSNO from amyl nitrite and n-butyl nitrite about 10-fold. Increasing concentrations of indomethacin inhibited both reactions in parallel. N-Acetyl-L-cysteine but not L-cysteine could substitute for GSH. It is concluded that rat liver microsomal GST catalyses the formation of GSNO from amyl nitrite and n-butyl nitrite. The activity of the MalNEt-stimulated microsomal GST is calculated to be about 17 units/mg of enzyme with the alkyl nitrites and about 16 units/mg of enzyme with CDNB as a substrate, assuming that 3% of microsomal protein is GST. These rates are comparable with those obtained for cytosolic GSTs. Thus microsomal GST may play a significant role in the metabolism of alkyl nitrites in biological membranes.

Alteration of liver glutathione S-transferase and protease activities by cobalt chloride treatment of rats.

Effects of cobalt chloride on liver glutathione S-transferase and protease activities were studied. When cobalt chloride (60 mg/kg) was given to rats, liver microsomal glutathione S-transferase and protease activities were significantly increased 24 hr after the injection, whereas glutathione peroxidase activity in microsomes was decreased. The increase in glutathione S-transferase by N-ethylmaleimide was similar to that of the control, indicating that the increase in the transferase activity by cobalt chloride is not due to a modification of the sulfhydryl group of the enzyme. Immunochemical analysis of the liver microsomes did not detect any proteolytic product of microsomal glutathione S-transferase. In puromycin- or actinomycin D-treated rats, an increase in the transferase activity caused by cobalt chloride treatment was depressed. Thus it was suggested that liver microsomal glutathione S-transferase is induced by cobalt chloride treatment, but not activated by limited proteolysis via microsomal protease.

Activation of microsomal glutathione S-transferase in tert-butyl hydroperoxide-induced oxidative stress of isolated rat liver.

The activation of microsomal glutathione S-transferase in oxidative stress was investigated by perfusing isolated rat liver with 1 mM tert-butyl hydroperoxide (t-BuOOH). When the isolated liver was perfused with t-BuOOH for 7 min and 10 min, microsomal, but not cytosolic, glutathione S-transferase activity was increased 1.3-fold and 1.7-fold, respectively, with a concomitant decrease in glutathione content. A dimer protein of microsomal glutathione S-transferase was also detected in the t-BuOOH-perfused liver. The increased microsomal glutathione S-transferase activity after perfusion with t-BuOOH was reversed by dithiothreitol, and the dimer protein of the transferase was also abolished. When the rats were pretreated with the antioxidant alpha-tocopherol or the iron chelator deferoxamine, the increases in microsomal glutathione S-transferase activity and lipid peroxidation caused by t-BuOOH perfusion of the isolated liver was prevented. Furthermore, the activation of microsomal GSH S-transferase by t-BuOOH in vitro was also inhibited by incubation of microsomes with alpha-tocopherol or deferoxamine. Thus it was confirmed that liver microsomal glutathione S-transferase is activated in the oxidative stress caused by t-BuOOH via thiol oxidation of the enzyme.

Increase in liver microsomal glutathione S-transferase activity by phenobarbital treatment of rats : Possible involvement of oxidative activation via cytochrome P450

The possible involvement of oxidative activation of liver microsomal glutathione (GSH) S-transferase by the cytochrome P450 system was investigated. When rats were given phenobarbital (PB) intraperitoneally for 3 days, liver microsomal GSH S-transferase activity was stimulated 1.3–1.4-fold and the effect of PB on the transferase was potentiated by combination with a catalase inhibitor, 3-amino-1,2,4-triazole. Immunoblotting of microsomal proteins from PB-treated rats with anti-microsomal GSH S-transferase antibody after SDS-PAGE showed the presence of a dimer of the transferase. When microsomal suspensions prepared from PB-treated rats were placed on ice without GSH, the microsomal GSH S-transferase activity gradually increased with time and reached 200% of the initial level at 3 hr when activation of the transferase by N-ethylmaleimide was lost. The time-dependent increase in GSH S-transferase activity in PB-treated microsomes was prevented by addition of 0.1 mM GSH. The increase in microsomal GSH S-transferase activity by NADPH was depressed by cytochrome P450 inhibitors such as SKF 525-A (2-diethylaminoethyl-2,2-diphenylvalerate), metyrapone or isoniazid in agreement with the concomitant decrease in generation of hydrogen peroxide in microsomes. These results indicate that the increase in GSH S-transferase activity in liver microsomes by PB treatment of rats is due to the oxidative modification of the enzyme by reactive oxygen species which are concomitantly increased following induction of cytochrome P450.

Organic Hydroperoxide-Induced Activation of Liver Microsomal Glutathione S-Transferase of Rats In Vitro

Neurochemical and behavioral investigations were made to assess the role of central dopaminergic systems in mouse locomotor activity and analgesia by spiradoline mesylate. Analgesic activities of the kappa-opioid-receptor agonists spiradoline and U-50488H were not altered by haloperidol or L-dopa, whereas morphine analgesia was enhanced by haloperidol but attenuated by L-dopa. Spiradoline decreased spontaneous locomotor activity in mice and inhibited methamphetamine- or morphine-induced locomotor activity. In contrast, morphine given alone increased locomotor activity and enhanced methamphetamine-induced locomotor activity. In a neurochemical study, spiradoline decreased the amounts of dopamine metabolites in the striatum, but did not alter them in the brainstem and cerebral cortex. Morphine increased the dopamine metabolite contents in all three brain regions tested. These results suggest that inhibition of the dopaminergic pathway in the brain by spiradoline may be involved in its suppression of locomotor activity, but not in its analgesia; whereas, stimulation of the dopaminergic pathway by morphine seems to function in both behaviors: enhancement of locomotor activity and inhibition of analgesia.

Non-Competitive Antagonism by Hirsuteine of Nicotinic Receptor-Mediated Dopamine Release from Rat Pheochromocytoma Cells

The effect of t-butyl hydroperoxide (t-BuOOH), cumene hydroperoxide (CuOOH) or linoleic acid hydroperoxide (linoleic-OOH) on liver microsomal glutathione S-transferase of rats was studied in vitro. When microsomes were incubated with either 100 microM t-BuOOH or 25 microM CuOOH, glutathione S-transferase activity was increased 1.5-fold; activity was further increased to 2.2-fold in the presence of small amounts of glutathione. The same amounts of dithiothreitol or cysteine did not enhance the t-BuOOH or CuOOH-induced increase in transferase activity. The transferase activity was also increased 1.4-fold by 10 microM linoleic-OOH plus 1 microM glutathione. The increase in microsomal glutathione S-transferase activity after treatment of microsomes with t-BuOOH in the presence of glutathione was completely reversed by addition of dithiothreitol, whereas the activation of the transferase caused by t-BuOOH in the absence of glutathione was not reversed. Although microsomal glutathione S-transferase also possesses glutathione peroxidase activity, only transferase activity was increased by t-BuOOH in either the presence or absence of glutathione. These data indicate that microsomal glutathione S-transferase is activated by organic hydroperoxides in either the absence or presence of small amounts of glutathione, suggesting an activation of the transferase by thiol oxidation of the cysteine residue.

Pesticides as activators of mouse liver microsomal glutathione S-transferase

Selected sulfhydryl-reactive pesticides activate mouse liver microsomal glutathione S-transferase (GST m) assayed in purified form free from cytosolic transferases (GST cs) with 1-chloro-2,4-dinitrobenzene as the substrate. Maximum activations were as follows: 1315% for N-ethylmaleimide (positive control) at 1 m M, 374% for chloranil at 0.01 m M, 272% for EPTC sulfoxide at 10 m M, 255% for captan at 0.1 m M, 228% for acrolein at 1 m M, and 152% for methyl isothiocyanate at 10 m M. Pesticides which showed little or no activation were alachlor, atrazine, tetramethrin, and tridiphane. The toxicological relevance of GST m activation was examined with the aforementioned activators plus EPTC and allyl alcohol. Experiments were conducted in vitro or in vivo with washed microsomes (which contain not only GST m but also some GST cs) and the post-mitochondrial supernatant fraction with its high GST c activity. These studies established that GST m, either unactivated or activated, was not a significant contributor to overall mouse liver GST activity.

Activation of rat liver microsomal glutathione S-transferase by hydrogen peroxide: Role for protein-dimer formation

The mechanism of oxygen radical-dependent activation of hepatic microsomal glutathione S-transferase by hydrogen peroxide was studied. Glutathione S-transferase activity in liver microsomes was increased 1.5-fold by incubation with 0.75 m m hydrogen peroxide at 37 °C for 10 min, and the increase in activity was reversed by incubation with dithiothreitol. Purified glutathione S-transferase was also activated by hydrogen peroxide after incubation at room temperature, and the increase in the activity was also reversed by dithiothreitol. Immunoblotting with anti-microsomal glutathione S-transferase antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hydrogen peroxide-treated microsomes or purified glutathione S-transferase revealed the presence of a glutathione S-transferase dimer. These results indicate that the hydrogen peroxide-dependent activation of the microsomal glutathione S-transferase is associated with the formation of a protein dimer.