Rat Liver Glutathione S-transferase Research Articles

Expression of glutathione S-transferase during rat liver development.

The ontogeny of rat liver glutathione S-transferase (EC 2.5.1.18) (GSTs) during foetal and postnatal development was investigated. The GSTs are dimers, the subunits of which belong to three multigene families, Alpha (subunits 1, 2, 8 and 10), Mu (subunits 3, 4, 6, 9 and 11) and Pi (subunit 7) [Mannervik, Alin, Guthenberg, Jennsson, Tahir, Warholm & Jörnvall (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7202-7206; Kispert, Meyer, Lalor, Coles & Ketterer (1989) Biochem. J. 260, 789-793]. There is considerable structural homology within each gene family, with the result that whereas reverse-phase h.p.l.c. successfully differentiates individual subunits, immunocytochemical and Northern-blotting analyses may only differentiate families. Enzymic activity, h.p.l.c. and Northern blotting indicated that expression of GST increased from very low levels at 12 days of foetal growth to substantial amounts at day 21. At birth, GST concentrations underwent a dramatic decline and remained low until 5-10 days post partum, after which they increased to adult levels. During the period under study, GST subunits underwent differential expression. The Mu family had a lower level of expression than the Alpha family, and, within the Alpha family, subunit 1 was more dominant in the adult than the foetus. Subunit 2 is the major form in the foetus. Most noteworthy were subunits 7 and 10, which were prominent in the foetus, but present at low levels post partum. Immunocytochemical analysis of the 17-day foetal and newborn rat livers showed marked differences in the distribution of GSTs in hepatocytes. In the 17-day foetal liver Pi greater than Alpha greater than Mu whereas in the newborns Alpha greater than Mu much greater than Pi. Erythropoietic cells were not stained for any of the three GST families. Steady-state mRNA concentrations in the foetus correlated with the relative transcription of the Alpha, Mu and Pi class genes. However, in those genes expressed post partum, namely the Alpha and Mu class, low transcriptional activity was associated with high concentrations of mRNA. This suggests that there is a switch from transcriptional control to post-transcriptional control at birth. GST 7-7 appears to be regulated predominantly by transcription throughout the period of liver development under observation.

Depletion of rat hepatic glutathione and inhibition of microsomal trans-2-enoyl-CoA reductase activity following administration of a dec-2-ynol and dec-2-ynoic acid

The effects of administration of dec-2-ynol and dec-2-ynoic acid on the hepatic glutathione (GSH) content and hepatic microsomal trans-2-enoyl-CoA reductase activity were examined in rat. Both compounds, when administered ip, caused a marked depletion of GSH levels and a corresponding inactivation of trans-2-enoyl-CoA reductase activity in both a time- and dose-dependent manner. The dec-2-ynoic acid caused greater hepatotoxicity than dec-2-ynol based on serum alanine transaminase activity. Based on the observations that (a) the alcohol did not interact with GSH in the presence or absence of cytosol, (b) the spectral manifestation of the interaction between GSH and the alcohol occurred only when NAD + was added to the reaction mixture containing the cytosol and reactants, and (c) a similar absorbance spectrum was obtained following the interaction between aldehyde and GSH, it was concluded that dec-2-ynol is converted to an electrophile, dec-2-ynal, which causes depletion of GSH. The decrease in GSH content following administration of the acid appears to be due to activation of the acid to the electrophile, dec-2-ynoyl CoA, which then interacts with GSH, resulting in its depletion, based on the in vitro observations that (a) the acid did not interact with GSH in the presence or absence of cytosol, and (b) the spectral manifestation of interaction between GSH and dec-2-ynoyl CoA occurred both nonenzymatically and enzymatically in the presence of rat liver glutathione S-transferase (Sigma). Bovine serum albumin stimulated the enzymatic reaction. Comparable to the effects on GSH were the effects of dec-2-ynol, dec-2-ynal, dec-2-ynoic acid, and dec-2-ynoyl CoA on the microsomal trans-2-enoyl-CoA reductase activity in vitro. While the alcohol had no effect on the enzyme activity, its electrophilic product, the aldehyde, was a potent inhibitor. Similarly, the acid did not inhibit the enzyme activity unless the acid was present at high concentration; however, its electrophilic product, the CoA thioester, was a very potent inhibitor at very low concentration.

Modulation of sulphobromophthalein excretion by ethacrynic acid.

1. Ethacrynic acid (EA), a phenoxyacetic acid diuretic, has similar effects to tienilic acid (TA) on rat liver glutathione S-transferase (GST) activity in vitro, using either 1-chloro-2,4-dinitrobenzene or sulphobromophthalein (BSP) as a substrate. EA inhibits the basic rat liver GST, with inhibition being greater with GST containing subunits 3 and 4 than with those containing subunits 1 and 2. 2. In vitro inhibitors of GST can inhibit biliary excretion of BSP in a perfused liver. 3. A single bolus dose of EA had no effect on BSP excretion from the isolated perfused rat liver, and this is most likely due to the rapid disappearance of EA from the perfusion media. Experiments using perfused rat liver indicated that a sustained high concentration of EA in the perfusion media has an inhibitory effect on the excretion of both unchanged and conjugated BSP. 4. A decrease in BSP excretion may not be an indicator of liver damage, but a consequence of GST inhibition.

S-(4-Bromo-2,3-dioxobutyl)glutathione: a new affinity label for the 4-4 isoenzyme of rat liver glutathione S-transferase

S-(4-Bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, has been synthesized and characterized by UV spectroscopy and thin-layer chromatography, as well as by bromide and primary amine analysis. Incubation of S-BDB-G (200 microM) with the 4-4 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. The kobs exhibits a nonlinear dependence on S-BDB-G concentration from 50 to 1000 microM, with a kmax of 0.078 min-1 and K1 = 66 microM. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, completely protects against inactivation by S-BDB-G. About 1.3 mol of [3H]S-BDB-G/mol of enzyme subunit is incorporated concomitant with 100% inactivation, whereas only 0.48 mol of reagent/mol of subunit is incorporated in the presence of S-hexylglutathione when activity is fully retained. Modified enzyme, prepared by incubating glutathione S-transferase with [3H]S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with NaBH4, carboxymethylated, and digested with trypsin. The tryptic digest was fractionated by reverse-phase high-performance liquid chromatography. Two radioactive peptides were identified: Lys82-His-Asn-Leu-X-Gly-Glu-Thr-Glu-Glu-Glu-Arg93, in which X is modified Cys86, and Leu109-Gln-Leu-Ala-Met-CmCys-Y-Ser-Pro-Asp-Phe-Glu-Arg121 , in which Y is modified Tyr115. Only the Lys82-Arg93 peptide was modified in the presence of S-hexylglutathione when the enzyme retained full activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystals of isoenzyme 3-3 of rat liver glutathione S-transferase with and without inhibitor

The isoenzyme 3-3 of rat liver glutathione S-transferase (GST 3-3) isolated from a baculovirus expression system has been crystallized with and without inhibitor. The crystals grown in the absence of an inhibitor belong to space group P2(1) with cell dimensions a = 119.7, b = 96.2, c = 136.7 A and beta = 103.3 degrees, and diffract to 3 A resolution. The crystals grown in the presence of an inhibitor belong to space group C2 with cell dimensions a = 88.3, b = 69.7, c = 81.4 A and beta = 105.3 degrees, and diffract to at least 2.5 A resolution. The inhibitor used is either methylmercury chloride or ethylmercury chloride; both are weak inhibitors.

Inhibition of hepatic glutathione- S-transferases by fatty acids and fatty acid esters

Micromolar concentrations of polyunsaturated fatty acids and ascorbate esters of saturated fatty acids were found to cause a marked inhibition of rat and mouse hepatic glutathione- S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene. Arachidonic acid was approximately 25 times more potent in inhibiting rat GST than palmitic acid which was the least effective. Both linoleic and arachidonic acids did not inhibit rat liver GST when ethacrynic acid was used as substrate while the reverse was true with 1,2-dichoro-4-nitrobenzene. In contrast, all the chemicals tested inhibited rat liver GST activity towards 4-nitropyridine N-oxide, indicating isozyme specificity.

Site-directed mutagenesis of glutathione S-transferase YaYa: Nonessential role of histidine in catalysis

A cDNA encoding a rat liver glutathione S-transferase Ya subunit has been expressed in Escherichia coli and the expressed enzyme purified to homogeneity. In order to examine the catalytic role of histidine in the glutathione S-transferase Ya homodimer, site-directed mutagenesis was used to replace all three histidine residues (at positions 8, 143, and 159) by other amino acid residues. The replacement of histidine 8 or histidine 143 with valine did not affect the 1-chloro-2,4-dinitrobenzene-conjugating activity nor the isomerase activity. However, the replacement of histidine with valine at position 159 produced the mutant GST which exhibited only partial activity. A greater decrease in catalytic activity was observed by histidine → tyrosine or histidine → lysine replacement at position 159. On the other hand, the histidine 159 → asparagine mutant retained full catalytic activity. Our results indicate that histidine residues in the Ya homodimer are not essential for catalytic activity. However, histidine 159 might be critical in maintaining the proper conformation of this enzyme since replacement of this amino acid by either lysine or tyrosine did result in significant loss of enzymatic activity.

Analysis of glutathione S-transferase-catalyzed S-alkylglutathione formation by high-performance liquid chromatography

Metabolism of alkyl halides and some organophosphorous compounds by glutathione S-transferases (EC 2.5.1.18) leads to formation of an S-alkylglutathione as a common product. We have developed an HPLC assay for formation of S-methylglutathione and S-ethylglutathione that is applicable to measuring enzyme activity toward a variety of xenobiotic substrates. The conjugates are derivatized with 1-fluoro-2,4-dinitrobenzene to form the corresponding N-2,4-dinitrophenyl derivatives, which are then separated by reverse-phase HPLC with gradient elution. The utility of the method is illustrated by the use of partially purified preparations of rat liver glutathione S-transferases and several prototypic substrates including iodomethane, iodoethane, dichlorvos, and methyl parathion. The limit of detection is about 50 pmol of N-(2,4-dinitrophenyl)- S-alkylglutathione. Advantages of the method over other assays of S-alkyl transferase activity are discussed.

Inhibition of rat liver glutathione S-transferase isoenzymes by peptides stabilized against degradation by gamma-glutamyl transpeptidase.

Inhibitors for glutathione S-transferase (GST) iso-enzymes from rat liver with high affinity for the glutathione-binding site (G-site) have been developed. In previous studies, a model was described for the G-site of GST (Adang, A. E. P., Brussee, J., van der Gen, A., and Mulder, G. J. (1990) Biochem. J. 269, 47-54) in terms of essential and nonessential interactions between groups in glutathione (GSH) and the G-site. Based on this model, compounds were designed that have high affinity for the G-site but cannot be conjugated. In the dipeptide gamma-L-glutamyl-D-aminoadipic acid (gamma-L-Glu-D-Aad), the L-cysteinylglycine moiety is replaced by D-aminoadipic acid. This dipeptide is an efficient competitive inhibitor (toward GSH) of mu class GST isoenzymes with Ki values of 34 microM for GST isoenzyme 3-3 and 8 microM for GST isoenzyme 4-4. Other GSH-dependent enzymes, such as gamma-glutamyl transpeptidase (gamma-GT), glutathione reductase, and glutathione peroxidase, were not inhibited by 1 mM of gamma-L-Glu-D-Aad. Inhibition is also highly stereospecific since gamma-L-Glu-L-Aad is only a poor inhibitor (Ki = 430 microM for GST 3-3). Gamma-L-Glutamyl-D-norleucine also had a much higher Ki value for GST 3-3. Thus, the presence of a delta-carboxylate group in D-Aad appears to be essential for a high affinity inhibitor. An additional hydrophobic group did not result in increased inhibitory potency. In a different approach, the gamma-L-glutamyl moiety in GSH was replaced by delta-L-aminoadipic acid; delta-L-Aad-L-Cys-Gly is an efficient cosubstrate analogue for GSTs with Km values comparable to GSH and Vmax values ranging from 0.24 to 57 mumol/min/mg for the different GSTs. The structures of the efficient inhibitor and the cosubstrate analogue were combined in delta-L-Aad-D-Aad, which had a Ki value of 68 microM with GST 3-3. In order to investigate their possible use in vivo studies, the degradation of gamma-L-Glu-D-Aad and delta-L-Aad-L-Cys-Gly by gamma-GT was investigated. The peptides showed no measurable hydrolysis rates under conditions where GSH was rapidly hydrolyzed. Thus, an efficient, mu class-specific GST inhibitor and a gamma-glutamyl-modified cosubstrate analogue of GSH were developed. Their gamma-GT stability offers the possibility to use these peptides in in vivo experiments.

Isozyme specificity in the conversion of hepoxilin A3 (HxA3) into a glutathionyl hepoxilin (HxA3-C) by the Yb2 subunit of rat liver glutathione S-transferase.

1-14C-Labeled hepoxillin A3 is transformed by a purified preparation of glutathione S-transferase in the presence of glutathione into a glutathionyl conjugate in which the glutathione is covalently coupled to the carbon 11 position of hepoxilin A3. We have termed the glutathione conjugate hepoxilin A3-C in keeping with the established nomenclature for glutathione conjugates in the leukotriene series. Using [3H]glutathione as cosubstrate, the kinetics of the reaction were followed. Among various rat liver glutathione S-transferase isozymes, a homodimer of the Yb2 subunit showed the best activity, while isozymes containing the Ya and Yc subunits showed marginal activity with hepoxilin A3 as substrate.

Synthesis and use of an isoform-specific affinity matrix in the purification of glutathione S-transferases from the housefly, Musca domestica (L.)

Glutathione may be linked to an agarose matrix which has been activated by treatment with epichlorhydrin. The resulting resin displayed group selectivity for the glutathione S-transferases of the housefly Musca domestics (L). The isoenzymes of low isoelectric point, which have little activity with substrates other than 1-chloro-2,4-dinitrobenzene, bound strongly to this matrix and were eluted with 10 mm glutathione at pH 7.4. On the other hand, the group of isoenzymes of higher isoelectric point, showing activity with other substrates such as 3,4-dichloronitrobenzene, did not bind. These isoenzymes did bind to a sulfobromophthalein-glutathione conjugate immobilized on agarose and could be eluted with 5 mm sulfobromophthalein at pH 7.4. The immobilized glutathione resin bound rat liver glutathione S-transferase subunits from all three molecular weight classes.

Evidence for Identifying Fatty Acids in Rat Liver Glutathione S-Transferase and Its Possible Involvement in the Secondary Structure1

A rat liver glutathione S-transferase isozyme (GST 3-3) has been purified to apparent electrophoretic homogeneity by procedures including Sephadex G-100, S-hexylglutathione-linked Sepharose, and CM-cellulose column chromatographies. The present study provides evidence for the existence of endogenous fatty acids in the purified GST 3-3 by gas-liquid chromatographic and mass-spectrometric analyses. The liver GST isozyme associated long chain fatty acids such as 14:0, 16:0, 18:0, and 18:1 and the molar ratio of fatty acids to the protein was estimated to be around 3.2. When the enzyme preparation was freed of fatty acids by a mild delipidization technique using Lipidex, the GST activity was significantly decreased. Computer analysis of the circular dichroism spectra revealed that rat liver GST 3-3 contained approximately 49% alpha-helix and 24% random coil. By delipidizing, the enzyme's alpha-helix was decreased to 4% and the random coil was in turn increased to 62%. The enzymatic and structural properties of the delipidized enzyme, however, could be restored to nearly the original levels by recombining with the fatty acids. These findings suggest that weakly bound fatty acids are responsible for the functional capacity of the GST by virtue of changing the protein conformation.

The glutathione-binding site in glutathione S-transferases. Investigation of the cysteinyl, glycyl and γ-glutamyl domains

The GSH-binding site of glutathione S-transferase (GST) isoenzymes was studied by investigating their substrate-specificity for three series of GSH analogues; further, a model of the interactions of GSH with the G-site is proposed. Twelve glycyl-modified GSH analogues, four ester derivatives of GSH and three cysteinyl-modified GSH analogues were synthesized and tested with purified forms of rat liver GST (1-1, 2-2, 3-3 and 4-4). The glycyl analogues exhibited spontaneous chemical reaction rates with 1-chloro-2,4-dinitrobenzene comparable with the GSH rate. In contrast, the enzymic rates (Vmax.) differed greatly, from less than 1 up to 140 mumol/min per mg; apparently, a reaction mechanism is followed that is very sensitive to substitutions at the glycyl domain. No correlation exists between the chemical rates and Vmax. values for the analogues. Analogues of GSH in which L-cysteine was replaced by D-cysteine, L-homocysteine or L-penicillamine showed little or no capacity to replace GSH as co-substrate for the GSTs. GSH monomethyl and monoethyl esters showed Vmax. values greater than the Vmax. measured with GSH: the Vmax. for the monoethyl ester of GSH and GST 3-3 was 5-fold that for GSH. The data obtained in this and previous studies [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724; Adang, Meyer, Brussee, van der Gen, Ketterer & Mulder (1989) Biochem. J. 264, 759-764] allow a model of the interactions of GSH in the G-site in GSTs to be postulated. The gamma-glutamyl site is the main binding determinant: the alpha-carboxylate group is obligatory, whereas shifting of the amino group and shortening of the peptide backbone only decreased kcat./Km. Furthermore, the GSTs appear to be very critical with respect to a correct orientation of the thiol group of the GSH analogue. The glycyl site is the least restrictive domain in the G-site of GSTs: amino acid analogues all showed Km values between 0.2 and 0.6 mM (that for GSH is 0.2-0.3 mM), but large differences in Vmax. exist. The glycyl carboxylate group is not essential for substrate recognition, since decarboxy analogues and ester derivatives showed high activities. The possible mechanisms for an increased Vmax. in some analogues are briefly discussed.

Expression in Escherichia coli of rat liver cytosolic glutathione S-transferase Yc cDNA

An expression plasmid, pKK-GTB2, containing the complete coding sequence of a rat liver glutathione S-transferase Yc subunit was constructed and expressed in Escherichia coli. The entire Yc cDNA sequence from plasmid pGTB42 (Telakowski-Hopskins et al., 1985, J. Biol. Chem. 260, 5820–5825) was amplified by the polymerase chain reaction, subcloned into modified expression vector A6316 (Schoner et al., 1986, Proc. Natl. Acad. Sci. USA 83, 8506–8510 and Linemeyer et al., 1987, Bio/Technology 5, 960–965) and transformed into E. coli strain AB1899. The colonies were screened by hybridization to pGTB42 and the production of Yc subunit was detected by immunoblot analysis. The purified recombinant Yc subunit was active in the conjugation and peroxidation reactions, and appeared homogeneous as judged by sodium dodecyl sulfate gel electrophoresis. Amino acid sequencing of the expressed Yc subunit revealed that about 40% of the expressed protein was blocked at the N-terminus. Approximately 25% of the sequenceable protein (15% of total protein) contained the initiation methionine residue at the amino terminus whereas the rest of the sequenceable protein had proline as the N-terminus. In contrast, only one molecular species with Pro as the first amino acid was identified when the inducer isopropyl-β- d-thiogalactopyranoside was omitted in the growth medium. Our observation indicated that under certain growth conditions, the enzymes responsible for protein maturation were not able to complete the processing of the overproduced recombinant Yc in E. coli.

The induction of specific rat liver glutathione S-transferase subunits under inadequate selenium nutrition causes an increase in prostaglandin F2 alpha formation.

We have reported previously that a dietary deficiency in selenium results in an increase in glutathione S-transferase (GST) activity of various rat tissues. In order to verify that the increased GST activity observed in a selenium deficiency results from increased synthesis of GST protein, cytosolic fractions of livers obtained from rats fed selenium-deficient and selenium-supplemented diets were analyzed by Western (protein) blots. Antisera raised against purified individual GST subunits (Ya, Yb, and Yc) were used to detect the corresponding subunits on the blots. The Ya subunit was induced 2.5-fold in the selenium-deficient state. The amount of Yc subunit also increased significantly (p less than 0.05) in selenium deficiency but not to the extent of the Ya subunit. The Yb subunit was not significantly affected by altered selenium nutritional status. A corresponding increase in poly(A) RNAs coding for the Ya and Yc subunits was also observed by Northern blot analysis. Transcriptional activity of GST YaYc genes was elevated by approximately 2-fold in purified nuclei isolated from selenium-deficient rat livers, which is sufficient to account for the increase in YaYc mRNA levels. Therefore, it appears that transcriptional activation of rat liver YaYc genes is the primary cause for the elevation of the corresponding gene products in the selenium-deficient state. Since the GSTs, especially the isozymes containing Ya subunit, have been implicated in the formation of prostaglandin (PG) F2 alpha, we investigated the effect of selenium deficiency on the PGF2 alpha-forming activity using a specific inhibitor of GSTs, S-decyl-GSH. In rats fed a nutritionally adequate diet, the activity inhibited by S-decyl-GSH accounted for at least half of the conversion of PGH2 to PGF2 alpha. During selenium deficiency, this GST-catalyzed activity was approximately doubled with no change in PGF2 alpha formation by other pathways, resulting in a 2-fold increase in overall synthesis of PGF2 alpha. These data strongly support a role of GSTs, especially those composed of the Ya size subunit, in the synthesis of PGF2 alpha from PGH2.

Isolation and characterization of the rat glutathione S-transferase Yb 1 subunit gene

We have isolated and characterized a rat liver glutathione S-transferase Yb 1 subunit gene. DNA sequence analysis of the Yb 1 subunit gene indicates that it comprises eight exons separated by seven introns and spans ~5.0 kb. The transcription initiation site has been mapped by primer extension experiments. Transcription begins at a guanine residue 29 nucleotides downstream from a “TATA” sequence. The DNA sequences of all exons and some introns share significant sequence identity with the corresponding exons and introns in the Yb 2 subunit gene characterized by Tu and co-workers [ J. Biol. Chem. 263, 11389–11395 (1988)]. The isolation and characterization of the glutathione S-transferase Yb 1 gene will allow for a detailed analysis of regulatory elements required for transcriptional regulation of this gene.

Construction, expression, and preliminary characterization of chimeric class μ glutathione S‐transferases with altered catalytic properties

An expression plasmid for isoenzyme 3-3 of rat liver glutathione S-transferase has been constructed from the cDNA clone pGTA/C44 and the pAS expression vector pMG27NS, and used for the efficient production of the enzyme in the Escherichia coli strain M5219. The plasmid has also been manipulated, through the use of synthetic linkers, to encode chimeric polypeptides in which short sequences of the closely related isoenzyme 4-4 have been substituted into the N-terminal and C-terminal variable domains of isoenzyme 3-3. The chimeric polypeptides designated 4(9)3(208), 3(209)4(8), and 4(9)3(200)4(8) are expressed with varying degrees of efficiency in E. coli. The active dimeric holoenzymes 3-3, (4(9)3(208]2, (3(209)4(8]2, and (4(9)3(200)4(8]2 can be isolated. The spectroscopic and kinetic properties of the chimeric enzymes are significantly different than the native enzyme.

Identification of rat liver glutathione S‐transferase Yb subunits by partial N‐terminal sequencing after electroblotting of proteins onto a polyvinylidene difluoride membrane from an analytical isoelectric focusing gel

Rat liver glutathione S-transferases were partially purified using S-hexyl glutathione affinity chromatography, followed by native isoelectric focusing employing a pH 7-11 or pH 3-10 gradient. Proteins were excised and eluted from the gel for determination of subunit composition using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In separate experiments, isoelectric focusing gels were equilibrated with a sodium dodecyl sulfate-containing buffer at high pH, and proteins on the gel were electroblotted onto a polyvinylidene difluoride membrane, utilizing graphite plates as electrodes. The membrane-bound proteins were visualized by Coomassie Brilliant Blue staining. The protein bands were then excised from the membrane and inserted into a gas phase sequenator for direct sequencing. N-Terminal sequences thus determined were compared with published cDNA sequences. The isoelectric points (pIs) and positions on the isoelectric focusing gel of Yb1Yb1, Yb1Yb2 and Yb2Yb2 subunits were determined. We have also located on the pH 3-10 focusing gel an N-terminal blocked glutathione S-transferase which has a molecular weight similar to Yb subunits.

Effect of diabetes and starvation on the activity of rat liver epoxide hydrolases, glutathione S-transferases and peroxisomal β-oxidation

The activities of peroxisomal β-oxidation, cytosolic and microsomal epoxide hydrolase as well as soluble glutathione S-transferases have been determined in the livers of alloxan- and streptozotocin-diabetic male Fischer-344 rats. Five, seven and ten days after initiation of diabetes serum glucose levels were elevated 3.6-, 5.7- to 6.2- and 6-fold, while the activities of peroxisomal β-oxidation and cytosolic epoxide hydrolase were elevated 1.5- and 2.5-fold, 1.4- and 2.7-fold and 1.3- and 2.0-fold, respectively. The activities of microsomal epoxide hydrolase and glutathione S-transferases were reduced to about 71% and 80% of controls. Application of 10 I.U./kg depot insulin twice a day for 10 consecutive days to alloxan-diabetic individuals approximately restored the initial glucose levels and enzyme activities except for peroxisomal β-oxidation. Starvation of Fischer-344 rats for 48 hours and 5 days similarly resulted in a 1.3-fold to 2.1-fold and 1.2- to 1.6-fold increase in peroxisomal β-oxidation and cytosolic epoxide hydrolase activity, respectively. Microsomal epoxide hydrolase was significantly decreased to 57% and 61% of control activity whereas glutathione S-transferase was only marginally reduced to 91% and 92%. Except for glutathione S-transferases initial enzyme activities were restored upon refeeding within 10 days. These results are similar to those obtained upon feeding of hypolipidemic compounds with peroxisome proliferating activity, and may indicate that high levels of free fatty acids or their metabolites which are known to accumulate in liver in both metabolic states may act as endogenous peroxisome proliferators.

Microsomal glutathione-dependent protection against lipid peroxidation acts through a factor other than glutathione peroxidase and glutathione S-transferase in rat liver

Ascorbate-Fe 3+-induced and NADPH-induced lipid peroxidation of rat liver microsomes were inhibited by glutathione (GSH). This inhibition was due to microsomal GSH-dependent factor. This factor was heat labile, and storage of microsomes at 4 °C for 1 week diminished the activity. GSH could not be substituted by other sulfhydryl compounds tested. Deoxycholate (1 m m) and bromosulfophthalein (0.1 m m) inhibited GSH-dependent protection but did not inhibit microsomal GSH peroxidase activity. Iodoacetate (10 m m) inhibited GSH-dependent protection but did not inhibit microsomal GSH S-transferase. N-Ethylmaleimide (0.1 m m) and oxidized glutathione (10 m m) inhibited GSH-dependent protection but activated microsomal GSH S-transferase activity. These results indicate the existence of a heat-labile, microsomal GSH-dependent protective factor against lipid peroxidation that acts through a factor other than GSH-peroxidase and GSH S-transferase.