Human liver microsomal glutathione transferase

Life Sciences, 1988

Glutathione S-transferases are a group of multifunctional isozymes that play a central role in the detoxification of hydrophobic xenobiotics with electrophilic centers (I). In this study we investigated the effects of in vitro lipid peroxidation on the

Chemico-Biological Interactions, 1990

A set of inhibitors for rat liver microsomal glutathione transferase have been characterized. These inhibitors (rose bengal, tributyltin acetate, Shexylglutathione, indomethacin, cibacron blue and bromosulphophtalein) all have I~ values in the 1-100 pM range. Their effects on the unactivated enzyme were compared to those on the N-ethylmaleimide-and trypsin-activated microsomal glutathione transferase. It was found that the I~0 values were decreased upon activation of the enzyme (5--20-fold), except for Shexylglutathione, where a slight increase was noted. Thus, the activated microsomal glutathione transferase is generally more sensitive to the effect of inhibitors than the unactivated enzyme. It was also noted that inhibitor potency can vary dramatically depending on the substrate used. The I~0 values for the N-ethylmaleimide-and trypsin-activated enzyme preparations are altered in a similar fashion compared to the unactivated enzyme.This finding indicates that these two alternative mechanisms of activation induce a similar type of change in the microsomal glutathione transferase.

Journal of Biological …, 1988

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 y-glutamyl-modified glutathione analogues, it was found that four (glutaryl-L-Cys-Gly, a-L-G~u-L-C~S-G~~, aD -Glu-L-Cys-Gly, and T-L-GIU-L-Cys-@-Ala) function as substrates. The kinetic parameters for three of these substrates (the ~-D-G~u-L-C~S-Gly analogue gave very low activity) were compared with those of GSH with both unactivated and the Nethylmaleimide-activated microsomal glutathione transferase. The a-~-G l u-~-C y~-G l y analogue is similar to GSH in that it has a higher kcat (6.9 uersus 0.6 s-') value with the activated enzyme compared with the unactivated enzyme but displays a high K , (6 uersus 11 mM) with both forms. Glutaryl-L-Cys-Gly, in

Biochemical Journal, 2001

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 # CO $ , 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 25 611.3 Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Eur. J. Biochem. 128, 243-248]. The microsomal GSTA1-Abbreviations

European Journal of Biochemistry, 1980

Rat liver microsomes were shown to catalyze the conjugation of 1-chloro-2,4-dinitrobenzene with glutathione and this activity has been characterized. It cannot be removed from the microsomes by washing or other procedures which release loosely bound material from membranes. The microsomal glutathione S-transferase can be activated up to eight fold by treatment with N-ethylmaleimide. This activation also affects the apparent Km of the enzyme(s) for both glutathione and 1-chloro-2,4-dinitrobenzene. Upon subcellular fractionation of the liver the N-ethylmaleimide-activateable glutathione S-transferase distributes in the same manner as a marker for the endoplasmic reticulum and unlike markers for the other organelles and for the cytoplasm. Treatment of microsomes with proteases revealed that the enzyme is at least partially exposed on the cytoplasmic surface of the endoplasmic reticulum. Finally, three inducers of drug-metabolizing systems-i.e. phenobarbital, methylcholanthrene, and trans-stilbene oxide-all increase the activity of the cytoplasmic glutathione S-transferases, but they do not affect the microsomal activity. These and other considerations indicate that the microsomal glutathione S-transferase(s) is distinct from the cytoplasmic enzymes catalyzing similar reactions. The microsomal enzyme is likely to be involved in drug metabolism and the possibility of activating it through attack on a sulfhydryl group may represent an important physiological response to certain xenobiotics.

Biochemical and Biophysical Research Communications, 2007

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.

FEBS letters, 1983

The amount and nature of glutathione transferases in rat liver microsomes were determined using immunological techniques. It was shown that cytosolic glutathione transferase subunits A plus C, and B plus L were present at levels of 2.4 k 0.6 and 1.5 f 0.1 pg/mg microsomal protein, respectively. These levels are IO-times higher than those for non-specific binding of cytosolic components judging from the distribution of lactate dehydrogenase, a cytosolic marker. The possibility that a portion of these glutathione transferases is functionally localized on the endoplasmic reticulum is discussed. A previously described microsomal glutathione transferase which is distinct from the cytosolic enzymes is present in an amount of 3 1 + 6 pg/mg microsomal protein. Glutathione transferase Microsomal Rat liver Drug metabolism

Journal of Biological Chemistry

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 y-glutamyl-modified glutathione analogues, it was found that four (glutaryl-L-Cys-Gly, a-L -G~u -L -C~S -G~~, a-D-Glu-L-Cys-Gly, and T-L-GIU-L-Cys-@-Ala) function as substrates. The kinetic parameters for three of these substrates (the ~-D -G~u -L -C~S -Gly analogue gave very low activity) were compared with those of GSH with both unactivated and the Nethylmaleimide-activated microsomal glutathione transferase. The a -~-G l u -~-C y~-G l y analogue is similar to GSH in that it has a higher kcat (6.9 uersus 0.6 s-') value with the activated enzyme compared with the unactivated enzyme but displays a high K , (6 uersus 11 mM) with both forms. Glutaryl-L-Cys-Gly, in contrast, exhibited a similar kc,, (8.9 versus 6.7 s-') with the N-ethylmaleimide-treated enzyme but retains a higher K , value (50 uersus 15 mM). Thus, the aamino 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/K, values, comparing the analogues to GSH and each other. It is estimated that the binding energy of the a-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.

Biochemical and Biophysical …, 1979

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 per fused with t-BuOOH for 7 min and 10 min, microsomal, but not cytosolic, glutathione S-transferase activ ity 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 a-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 a-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.

The Biochemical journal, 1997

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 ...

Glutathione S-transferases (GSTs) are isoenzymes occurring in the cytoplasm and as integral membrane proteins. In addition to their role in drug metabolism by conjugating electrophilic and lipophilic compounds with glutathione (GSH), these enzymes display multiple functions in cells, including antioxidant action. It has been generalized that reactive oxygen species (ROS) inhibit cytosolic GSTs and activate microsomal GSTs; some evidence shows, however, that different ROS-generating systems can inhibit microsomal GST activity. We therefore tested the effect of Fe 3+ /ascorbate, another ROS-generating system, on cytosolic and microsomal GST activities from rat liver cytosol and microsomes, respectively, and compared it to that of hydrogen peroxide (H 2 O 2 ). We found that, while both agents displayed similar inhibitory effects on cytosolic GST activity, they promoted opposite effects on microsomal GST activity. Using specific antioxidant enzymes, we corroborated that the effect of Fe 3+ /ascorbate involves generation of O 2 À without dismutation into H 2 O 2 . Since these ROS have physicochemical properties and redox potentials that are very distinct, their reactivity is different, and their oxidative action is likely to have different targets. We discuss how these properties are related with the oxidative potency of ROS, especially those of O 2 À and H 2 O 2 .

Biochemical Journal, 1995

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 1chloro-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-vectortreated 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 ,#-D-thiogalactopyranoside (IPTG; 1 mM) induced the

Archives of Biochemistry and Biophysics, 1999

In order to elucidate the protective role of glutathione S-transferases (GSTs) against oxidative stress, we have investigated the kinetic properties of the human ␣-class GSTs, hGSTA1-1 and hGSTA2-2, toward physiologically relevant hydroperoxides and have studied the role of these enzymes in glutathione (GSH)-dependent reduction of these hydroperoxides in human liver. We have cloned hGSTA1-1 and hGSTA2-2 from a human lung cDNA library and expressed both in Escherichia coli. Both isozymes had remarkably high peroxidase activity toward fatty acid hydroperoxides, phospholipid hydroperoxides, and cumene hydroperoxide. In general, the activity of hGSTA2-2 was higher than that of hGSTA1-1 toward these substrates. For example, the catalytic efficiency (k cat /K m) of hGSTA1-1 for phosphatidylcholine (PC) hydroperoxide and phosphatidylethanolamine (PE) hydroperoxide was found to be 181.3 and 199.6 s ؊1 mM ؊1 , respectively, while the catalytic efficiency of hGSTA2-2 for PC-hydroperoxide and PE-hydroperoxide was 317.5 and 353 s ؊1 mM ؊1 , respectively. Immunotitration studies with human liver extracts showed that the antibodies against human ␣-class GSTs immunoprecipitated about 55 and 75% of glutathione peroxidase (GPx) activity of human liver toward PC-hydroperoxide and cumene hydroperoxide, respectively. GPx activity was not immunoprecipitated by the same antibodies from human erythrocyte hemolysates. These results show that the ␣-class GSTs contribute a major portion of GPx activity toward lipid hydroperoxides in human liver. Our results also suggest that GSTs may be involved in the reduction of 5-hydroperoxyeicosatetrae-noic acid, an important intermediate in the 5-lipoxygenase pathway.

European Journal of Biochemistry, 2001

Toxicology and Applied Pharmacology, 2002

Glutathione S-transferases (GST) are multifunctional proteins. α class GSTs are known to catalyze glutathione peroxidase reactions, in addition to their major activity, i.e., conjugation of electrophiles to glutathione. In the present work, the contribution of rat and mouse α class GSTs to glutathione-dependent reduction of phospholipid hydroperoxides has been studied., Results of these studies indicate that the α class GST fraction, which consists of three isoforms, has glutathione peroxidase activity toward phospholipid hydroperoxides residing in biological membranes, without the need of prior phospholipase C action. Immunotitration studies using antibodies specific to the α class GSTs, GSTA1-1, GSTA2-2, and GSTA3-3, indicate that these GST isozymes account for approximately half of the glutathione peroxidase activity toward phospholipid hydroperoxides present in the 28,000g supernatant fractions of rat and mouse liver extracts. GSTs contribute proportionally lesser fraction of this activity in other tissues in which α class GSTs are less prevalent. In mice, the contribution of α class GSTs to the overall glutathione peroxidase activity is indistinguishable in wild-type mice and knockout mice lacking the major selenoenzyme, glutathione peroxidase 1, an enzyme that does not act on intact phospholipid hydroperoxides. These results are consistent with our previous studies on human α class GSTs (Yang, et al. J. Biol. Chem. 276, 19220–19230, 2001) and demonstrate that α class GSTs are of physiological importance, not only in the conjugative detoxification of electrophiles, but are also an essential component of cellular antioxidant defense mechanisms.