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.     

Activation of rat liver microsomal glutathione S-transferase by reduced oxygen species

The effect of enzymatically generated reduced oxygen metabolites on the activity of hepatic microsomal glutathione S-transferase activity was studied to explore possible physiological regulatory mechanisms of the enzyme. Noradrenaline and the microsomal cytochrome P-450-dependent monooxygenase system were used to generate reduced oxygen species. When noradrenaline (greater than 0.1 mM) was incubated with rat liver microsomes in phosphate buffer (pH 7.4), an increase in microsomal glutathione S-transferase activity was observed, and this activation was potentiated in the presence of a NADPH-generating system; the glutathione S-transferase activity was increased to 180% of the control with 1 mM noradrenaline and to 400% with both noradrenaline and NADPH. Superoxide dismutase and catalase inhibited partially the noradrenaline-dependent activation of the enzyme. In the presence of dithiothreitol and glutathione, the activation of the glutathione S-transferase by noradrenaline, with or without NADPH, was not observed. In addition, the activation of glutathione S-transferase activity by noradrenaline and glutathione disulfide was not additive when both compounds were incubated together. These results indicate that the microsomal glutathione S-transferase is activated by reduced oxygen species, such as superoxide anion and hydrogen peroxide. Thus, metabolic processes that generate high concentrations of reduced oxygen species may activate the microsomal glutathione S-transferase, presumably by the oxidation of the sulfhydryl group of the enzyme, and this increased catalytic activity may help protect cells from oxidant-induced damage.     

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 (gamma-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 1mM melphalan with 5mM GSH at 37 degrees C in aqueous phosphate buffer for 1h 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.     

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 (gamma-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 1mM CHB with 5 mM GSH at 37 degrees 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-[2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG2) 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 (CHBOH2), 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 CHBSG2, 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 activity by sulfhydryl reagents.

Rat liver microsomes exhibit glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as the second substrate. This activity can be stimulated 8-fold by treatment of the microsomes with N-ethylmaleimide and 4-fold with iodoacetamide. The corresponding glutathione S-transferase activity of the supernatant fraction is not affected by such treatment. These findings suggest that rat liver microsomes contain glutathione S-transferase distinct from those found in the cytoplasmic and that the microsomal transferase can be activated by modification of microsomal sulfhydryl group(s).     

Activation of rat liver microsomal glutathione transferase by limited proteolysis.

The activity of rat liver microsomal glutathione transferase is increased by limited tryptic proteolysis; the membrane-bound and purified forms of the enzyme are activated about 5- and 10-fold respectively. The cleavage sites that correlate with this activation were determined by amino acid sequence analysis to be located after Lys-4 and Lys-41. Differences in the relative extent of cleavage at these two sites did not consistently affect the degree of activation. Thus the data support the conclusion that cleavage at either site results in activation. The trypsin-activated enzyme was compared with the form activated with N-ethylmaleimide, which modifies Cys-49. These two differently activated forms were found to have similar kinetic parameters, which differ from those of the unactivated enzyme. The relatedness of the two types of activation is also demonstrated by the observation that microsomal glutathione transferase fully activated by N-ethylmaleimide is virtually resistant to further activation by trypsin. This is the case despite the fact that the N-ethylmaleimide-activated enzyme is much more susceptible to trypsin cleavage at Lys-41 than is the untreated enzyme. The latter observation indicates that activation with N-ethylmaleimide is accompanied by a conformational change involving Lys-41.     

Activation of the microsomal glutathione S-transferase by metabolites of alpha-methyldopa

Rat liver microsomes contain a membrane-bound GSH S-transferase (GSH-tr), an enzyme that is involved in the detoxication of xenobiotics. Also located on rat liver microsomes is the cytochrome P450 system, an enzyme complex that catalyzes the conversion of several xenobiotics into reactive intermediates. In this study, it was demonstrated that reactive products from alpha-methyldopa formed by the cytochrome P450 system are able to stimulate microsomal GSH-tr. Also, products formed from alpha-methyldopa that are generated by H2O2-horseradish peroxidase and tyrosinase are able to stimulate the activity of microsomal GSH-tr. GSH was able to prevent the activation of microsomal GSH-tr. Our results indicate that the ortho-quinone or semi-ortho-quinone radical of alpha-methyldopa is responsible for the stimulation of microsomal GSH-tr, probably via arylation of the free sulfhydryl group of microsomal GSH-tr. This conclusion was supported by the observation that 4-methyl-ortho-quinone itself was able to stimulate microsomal GSH-tr via sulfhydryl arylation. Our results are in conformity with the hypothesis that reactive products formed by the cytochrome P450 complex are able to stimulate microsomal GSH-tr and possibly in this way enhance their detoxication.     

Regulation of rat liver microsomal glutathione S-transferase activity by thiol/disulfide exchange

The regulation of purified glutathione S-transferase from rat liver microsomes was studied by examining the effects of various sulfhydryl reagents on enzyme activity with 1-chloro-2,4-dinitrobenzene as the substrate. Diamide (4 mM), cystamine (5 mM), and N-ethylmaleimide (1 mM) increased the microsomal glutathione S-transferase activity by 3-, 2-, and 10-fold, respectively, in absence of glutathione; glutathione disulfide had no effect. In presence of glutathione, microsomal glutathione S-transferase activity was increased 10-fold by diamide (0.5 mM), but the activation of the transferase by N-ethylmaleimide or cystamine was only slightly affected by presence of glutathione. The activation of microsomal glutathione S-transferase by diamide or cystamine was reversed by the addition of dithiothreitol. Glutathione disulfide increased microsomal glutathione S-transferase activity only when membrane-bound enzyme was used. These results indicate that microsomal glutathione S-transferase activity may be regulated by reversible thiol/disulfide exchange and that mixed disulfide formation of the microsomal glutathione S-transferase with glutathione disulfide may be catalyzed enzymatically in vivo.     

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.     

Increase in glutathione disulfide level regulates the activity of microsomal glutathione S-transferase in rat liver

Glutathione disulfide stimulates the activity of rat liver microsomal glutathione S-transferase 2-fold after incubation at 25 degrees C for 10 min. When the microsomes were incubated with the disulfide for over 20 min, the transferase activity increased to the same extent as in the case of N-ethylmaleimide (6-fold). Even in the presence of reduced glutathione, some enhancement of the transferase activity was observed. The data presented here are evidence that increase in glutathione disulfide level, e.g. by lipid peroxidation, on endoplasmic reticulum causes the upregulation of microsomal glutathione S-transferase activity.     

Different contribution of rat liver microsomal pigments in the formation of superoxide anions and hydrogen peroxide during development.

Liver microsomes of adult rats produce, by an NADPH-dependent pathway, O₂^? radicals, as detected by the epinephrine cooxidation to adrenochrome (24.8 nmol/min/mg of protein). This production has also been measured during liver development (from 1 to 20 days after birth) and correlated to the enzyme content (NADPH-cytochrome c reductase, cytochrome b₅, and cytochrome P-450), with the aim of establishing the level at which Superoxide radicals are formed in the electron transport system. At 1 day the adrenochrome formation and the activity of NADPH-cytochrome c reductase are about 50 and 40% of those of the adult, respectively, whereas those of cytochromes b₅ and P-450 are approximately 10%. After 20 days of development cytochrome b₅ and the dehydrogenase reach the adult level, while cytochrome P-450 is about 80%. At this age O₂^? radicals have a 30% increment and reach only 60% of those of the adult; H₂O₂ production is also 60% and the N-demethylation of aminopyrine is only 30%. Thus, at birth the formation of O₂^? radicals is almost entirely dependent on the activity of the flavoprotein. The close correlation between the slight increase in the demethylase activity and adrenochrome formation from 1 to 20 days suggests that a portion of O₂^? radicals produced by the NADPH-dependent electron transfer is directly involved in the mixed function oxidation. Since about 50% of the radicals are formed at the flavoprotein level, these results indicate that in the adult liver the remaining amount may be generated at the level of cytochrome P-450.     

Inhibition of rat liver glutathione S-transferase isoenzymes by acrolein

The in vitro effect of the toxin and teratogen, acrolein, on the fetal rat liver glutathione S-transferase isoenzyme, YcYfetus, was investigated and compared with acrolein's effect on some of the adult rat liver glutathione S-transferase isoenzymes. Acrolein was found to inhibit all the isoenzymes investigated and double-reciprocal plots suggest that inhibition is either noncompetitive or mixed-type noncompetitive. It is therefore attractive to suggest that should a similar situation arise in vivo, it may provide one mechanism for the teratogenicity of acrolein.     

Effect of fraction V powder from bovine serum on rat liver microsomal glutathione S-transferase activity

When bovine serum fraction V powder, which contains 98-99% albumin, is incubated with rat liver microsomes, the glutathione S-transferase activity is stimulated 3-fold. The fraction V stimulates the transferase activity even in microsomes pretreated with N-ethylmaleimide. There is no such stimulating activity in protein fractions other than fraction V. The corresponding cytosolic enzyme activity is not changed by fraction V powder or by N-ethylmaleimide. Neither treatment with charcoal nor heating in boiling water for 5 min can remove the stimulating activity from fraction V.     

Radiation inactivation of microsomal glutathione S-transferase

Radiation inactivation analysis was used to determine the target size of rat liver microsomal glutathione S-transferase both in situ and following purification. When Tris-HCl-washed microsomes were irradiated, there was a 1.5-2.0-fold increase in enzymatic activity over the first 3-6 megarads followed by a decrease in enzymatic activity. Above 48 megarads the radiation inactivation curve of the Tris-HCl-washed microsomes was described by a monoexponential function which gave a target size of 48 kDa. The enzymatic activity of the microsomal enzyme was selectively increased by treating the Tris-HCl-washed microsomes either with N-ethylmaleimide or washing the microsomes with small unilamellar vesicles made from phosphatidylcholine. The inactivation curves obtained with both types of treated microsomes were simple monoexponential decays in enzymatic activity with target sizes of 46 kDa (N-ethylmaleimide) and 44 kDa (unilamellar vesicles). The microsomal enzyme was detergent solubilized and purified. The Mr value of the purified protein was 15,500 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). These data suggest that the functional unit of the microsomal form of glutathione S-transferase in situ is a trimer. The target size of the purified enzyme solubilized in Triton X-100 was 85 kDa, and no increase in activity was observed at the lower radiation doses. The increase in the target size of the purified enzyme could not be ascribed solely to the presence of the detergent. This result suggests that the microsomal form of this enzyme can exist as catalytically active oligomers of different sizes depending on its environment.     

Activation and inhibition of microsomal glutathione transferase from mouse liver.

Mouse liver microsomal glutathione transferase was purified in an N-ethylmaleimide-activated as well as an unactivated form. The enzyme had a molecular mass of 17 kDa and a pI of 8.8. It showed cross-reactivity with antibodies raised against rat liver microsomal glutathione transferase, but not with any of the available antisera raised against cytosolic glutathione transferases. The fully N-ethylmaleimide-activated enzyme could be further activated 1.5-fold by inclusion of 1 microM-bromosulphophthalein in the assay system. The latter effect was reversible, which was not the case for the N-ethylmaleimide activation. At 20 microM-bromosulphophthalein the activated microsomal glutathione transferase was strongly inhibited, while the unactivated form was activated 2.5-fold. Inhibitors of the microsomal glutathione transferase from mouse liver showed either about the same I50 values for the activated and the unactivated form of the enzyme, or significantly lower I50 values for the activated form compared with the unactivated form. The low I50 values and the steep slope of the activity-versus-inhibitor-concentration curves for the latter group of inhibitors tested on the activated enzyme indicate a co-operative effect involving conversion of activated enzyme into the unactivated form, as well as conventional inhibition of the enzyme.     

Effect of superoxide dismutase on hydroxylase activity and hydrogen peroxide formation in anthranilamide hydroxylation by a rat liver microsomal monooxygenase system

Anthranilamide was slightly hydroxylated by a reconstituted rat liver microsomal monooxygenase system with NADPH, but a large amount of hydrogen peroxide was formed with a consumption of NADPH during the reaction. Superoxide dismutase stimulated the hydroxylation by depressing the hydrogen peroxide formation, in that there was a reverse correlation between the two effects due to the dismutase. In addition, a trace of 3-hydroxyanthranilamide, one of the products, not only stimulated NADPH-dependent hydrogen peroxide formation via NADPH-cytochrome c (P-450) reductase, but also inhibited the reduction of cytochrome P-450 by NADPH in the reconstituted system. These effects of 3-hydroxyanthranilamide were also diminished by superoxide dismutase.     

Studies of endogenous inhibitors of microsomal glutathione S-transferase.

Glutathione S-transferase is present in rat liver microsomal fraction, but its activity is low relative to the transferase activity present in the soluble fraction of the hepatocyte. We have found, however, that the activity of microsomal glutathione S-transferase is increased 5-fold after treatment with small unilamellar vesicles made from phosphatidylcholine. The increase in activity is due to the removal of an inhibitor of the enzyme from the microsomal membrane. The inhibitor is present in the organic layer of a washed Folch extract of the microsomal fraction. When this fraction of the microsomal extract is reconstituted in the form of small unilamellar vesicles, it inhibits microsomal glutathione S-transferase that had been activated by prior treatment with small unilamellar vesicles of pure phosphatidylcholine, but does not affect the activity of unactivated microsomal glutathione S-transferase. The inhibitor did not seem to be formed during the isolation of the microsomal fraction, and hence may be a physiological regulator of microsomal glutathione S-transferase. In this regard, both free fatty acid (palmitate) and lysophosphatidylcholine were shown to inhibit the enzyme reversibly. The results indicate that the activity of microsomal glutathione S-transferase is far greater than appreciated until now, and that this form of the enzyme may be an important factor in the hepatic metabolism of toxic electrophiles.     

Inhibition of cytosolic glutathione S-transferase activity from rat liver by copper

H(2)O(2) inactivation of particular GST isoforms has been reported, with no information regarding the overall effect of other ROS on cytosolic GST activity. The present work describes the inactivation of total cytosolic GST activity from liver rats by the oxygen radical-generating system Cu(2+)/ascorbate. We have previously shown that this system may change some enzymatic activities of thiol proteins through two mechanisms: ROS-induced oxidation and non-specific Cu(2+) binding to protein thiol groups. In the present study, we show that nanomolar Cu(2+) in the absence of ascorbate did not modify total cytosolic GST activity; the same concentrations of Cu(2+) in the presence of ascorbate, however, inhibited this activity. Micromolar Cu(2+) in either the absence or presence of ascorbate inhibited cytosolic GST activity. Kinetic studies show that GSH but no 1-chloro-2,4-dinitrobenzene prevent the inhibition on cytosolic GST induced by micromolar Cu(2+) either in the absence or presence of ascorbate. On the other hand, NEM and mersalyl acid, both thiol-alkylating agents, inhibited GST activity with differential reactivity in a dose-dependent manner. Taken together, these results suggest that an inhibitory Cu(2+)-binding effect is likely to be negligible on the overall inhibition of cytosolic GST activity observed by the Cu(2+)/ascorbate system. We discuss how modification of GST-thiol groups is related to the inhibition of cytosolic GST activity.     

Glycerophosphate-dependent hydrogen peroxide production by rat liver mitochondria

We studied the extent to which hormonally-induced mitochondrial glycerophosphate dehydrogenase (mGPDH) activity contributes to the supply of reducing equivalents to the mitochondrial respiratory chain in the rat liver. The activity of glycerophosphate oxidase was compared with those of NADH oxidase and/or succinate oxidase. It was found that triiodothyronine-activated mGPDH represents almost the same capacity for the saturation of the respiratory chain as Complex II. Furthermore, the increase of mGPDH activity induced by triiodothyronine correlated with an increase of capacity for glycerophosphate-dependent hydrogen peroxide production. As a result of hormonal treatment, a 3-fold increase in glycerophosphate-dependent hydrogen peroxide production by liver mitochondria was detected by polarographic and luminometric measurements.