Chemical modification of human placental glutathione transferase by pyridoxal 5'-phosphate.

Incubation of GST pi from human placenta with 8 mM PLP resulted in a rapid loss of activity during the first 10 min, concomitant with a Schiff base formation. This inactivation was probably due to the formation of a reversible adduct between PLP and the enzyme. After sodium borohydride treatment this adduct was reduced and stabilized. Stoichiometry and peptide isolation studies showed that three lysine residues were modified during reaction of GST and PLP. Protection of the enzyme against inactivation was achieved in the presence of 4 mM GSH suggesting that at least one lysyl residue is associated with the substrate binding site. Peptide mapping by digesting the enzyme with trypsin revealed that lysine shielded by GSH is Lys-127. Our results suggest that this residue may play an important role in enzymatic activity.     

Lipoxygenase-another pathway for glutathione conjugation of xenobiotics: A study with human term placental lipoxygenase and ethacrynic acid.

In this study, we examined the ability of human term placental lipoxygenase (HTPLO) to catalyze glutathione (GSH) conjugate formation from ethacrynic acid (EA) in the presence of linoleic acid (LA) and GSH. HTPLO purified by affinity chromatography was used in all the experiments. The results indicate that the process of EA-SG is enzymatic in nature. The reaction shows dependence on pH, the enzyme, and the concentration of GSH, LA, and EA. The optimal assay conditions to observe a maximal rate of EA-SG formation required the presence of 0.3 mM LA, 0.2 mM EA, 2.0 mM GSH, and approximately 300 microg HTPLO in the reaction medium buffered at pH 9.0. Under the experimental conditions employed, the reaction exhibited K(m) values of 1.1 mM, 200 microM, and 130 microM for GSH, LA, and EA, respectively. The estimated specific activity of HTPLO-catalyzed EA-GS formation was approximately 4.4 +/- 0.4 micromol/min/mg protein. This rate is more than twofold greater than the rate noted for the reaction mediated by the purified human term placental glutathione transferase. Under physiologically relevant conditions (20 microM LA, 2.0 mM GSH, at pH 7.4), HTPLO produced EA-SG at 56% of the maximal rate noted under optimal assay conditions. Nordihydroguaiaretic acid, the classical inhibitor of different lipoxygenases, significantly blocked the reaction. It is proposed that free radicals are involved in the process of EA-SG formation by HTPLO. The evidence gathered in this in vitro study suggests for the first time that lipoxygenase present in the human term placenta is capable of EA-SG formation.     

Purification of a glutathione S-transferase and a glutathione conjugate-specific dehydrogenase involved in isoprene metabolism in Rhodococcus sp. strain AD45

A glutathione S-transferase (GST) with activity toward 1, 2-epoxy-2-methyl-3-butene (isoprene monoxide) and cis-1, 2-dichloroepoxyethane was purified from the isoprene-utilizing bacterium Rhodococcus sp. strain AD45. The homodimeric enzyme (two subunits of 27 kDa each) catalyzed the glutathione (GSH)-dependent ring opening of various epoxides. At 5 mM GSH, the enzyme followed Michaelis-Menten kinetics for isoprene monoxide and cis-1, 2-dichloroepoxyethane, with Vmax values of 66 and 2.4 micromol min-1 mg of protein-1 and Km values of 0.3 and 0.1 mM for isoprene monoxide and cis-1,2-dichloroepoxyethane, respectively. Activities increased linearly with the GSH concentration up to 25 mM. 1H nuclear magnetic resonance spectroscopy showed that the product of GSH conjugation to isoprene monoxide was 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB). Thus, nucleophilic attack of GSH occurred on the tertiary carbon atom of the epoxide ring. HGMB was further converted by an NAD+-dependent dehydrogenase, and this enzyme was also purified from isoprene-grown cells. The homodimeric enzyme (two subunits of 25 kDa each) showed a high activity for HGMB, whereas simple primary and secondary alcohols were not oxidized. The enzyme catalyzed the sequential oxidation of the alcohol function to the corresponding aldehyde and carboxylic acid and followed Michaelis-Menten kinetics with respect to NAD+ and HGMB. The results suggest that the initial steps in isoprene metabolism are a monooxygenase-catalyzed conversion to isoprene monoxide, a GST-catalyzed conjugation to HGMB, and a dehydrogenase-catalyzed two-step oxidation to 2-glutathionyl-2-methyl-3-butenoic acid.     

The enzymatic defluorination of fluoroacetate in mouse liver cytosol: the separation of defluorination activity from several glutathione S-transferases of mouse liver

The liberation of free fluoride ion from fluoroacetate (FAc) proceeds as an enzyme-catalyzed dehalogenation reaction in the soluble fractions of several organs of the CFW Swiss mouse. Liver contained the highest FAc defluorinating activity. The enzyme activity in other organs decreased in the order kidney greater than lung greater than heart greater than testes. No activity was detected in the brain. Experiments were designed to characterize and identify the enzyme species responsible for FAc metabolism in liver. Enzyme activity was dependent on the concentration of glutathione (GSH) in the assay mixture, with maximal activity occurring above 5 mM. The dehalogenation of FAc had an apparent Km of 7.0 mM when measured in the presence of a saturating concentration of GSH. An increase in the pH of the assay mixture enhanced fluoride release in both phosphate and borate buffer. The defluorination activity was reduced to negligible levels when stored for 24 h at 4 degrees C. The addition of either GSH, dithiothreitol, or 2-mercaptoethanol increased stability, with the latter providing protection for greater than 150 h at a concentration of 15 mM. DEAE anion-exchange chromatography separated the defluorinating activity from 90% of the soluble GSH S-transferase activity measured with 1-chloro-2,4-dinitrobenzene. FAc defluorination activity did not bind to a GSH affinity column which selectively separates it from a group of anionic GSH S-transferases. The GSH-dependent enzyme which dehalogenates FAc has unique properties and can be separated from the liver GSH S-transferases previously described in the literature.     

Glutathione mediated regulation of oligomeric structure and functional activity of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> glutathione S-transferase

Background  

In contrast to many other organisms, the malarial parasite Plasmodium falciparum possesses only one typical glutathione S-transferase. This enzyme, PfGST, cannot be assigned to any of the known GST classes and represents a most interesting target for antimalarial drug development. The PfGST under native conditions forms non-covalently linked higher aggregates with major population (~98%) being tetramer. However, in the presence of 2 mM GSH, a dimer of PfGST is observed. Recently reported study on binding and catalytic properties of PfGST indicated a GSH dependent low-high affinity transition with simultaneous binding of two GSH molecules to PfGST dimer suggesting that GSH binds to low affinity inactive enzyme dimer converting it to high affinity functionally active dimer. In order to understand the role of GSH in tetramer-dimer transition of PfGST as well as in modulation of functional activity of the enzyme, detailed structural, functional and stability studies on recombinant PfGST in the presence and absence of GSH were carried out.     

Characterization of methylation of rat liver cytosolic glutathione S-transferases by using reverse-phase h.p.l.c. and chromatofocusing.

Glutathione S-transferase (GST) subunits in rat liver cytosol were separated by reverse-phase h.p.l.c.; five major proteins were isolated and identified as subunits 1, 2, 3, 4 and 8. F.p.l.c. chromatofocusing resolved the affinity-purified GST pool into nine different isoenzymes. The five basic (Alpha class) dimeric peaks of GST activity were 1-1, 1-2a, 1-2b, 2-2a and 2-2b. Reverse-phase h.p.l.c. analysis revealed that subunit 8 was also present in the protein peaks designated 1-1, 1-2a and 1-2b. The four neutral (Mu class) isoenzymes were 3-3, 3-4, 3-6 and 4-4. The GST pool was methylated in vitro before reverse-phase h.p.l.c. or f.p.l.c. chromatofocusing. Chromatofocusing indicated that the Mu class isoforms (3-3, 3-4 and 4-4) were the primary GSTs methylated, and h.p.l.c. analysis confirmed that subunits 3 and 4 were the major methyl-accepting GST subunits. The addition of calmodulin stimulated the methylation in vitro of GST isoenzymes 3-3, 3-4 and 4-4 by 3.0-, 7.5- and 9.9-fold respectively. Reverse-phase h.p.l.c. also indicated that only the methylation of GST subunits 3 and 4 was stimulated by calmodulin. Basic GST isoenzymes were minimally methylated and the methylation was not enhanced by calmodulin. Investigation of the time course of methylation of GST subunits 3 and 4 indicated that at incubation times less than 4 h the methylation of both Mu class subunits was stimulated by calmodulin, and that under such conditions subunit 4 was the preferred substrate. In contrast, there was essentially no calmodulin-stimulated methylation at incubation times of 4 or 6 h, and the methylation of subunit 3 was predominant. Kinetic parameters at 2 h of incubation were determined in the presence and in the absence of calmodulin. The addition of calmodulin doubled the Vmax. for methylation of both subunits 3 and 4 and decreased the Km of subunit 4 for S-adenosyl-L-methionine 3.6-fold. Finally, methylation was substoichiometric and after 6 h of incubation ranged from 2.8 to 7.6% on a mole-to-mole basis for subunits 4 and 3 respectively.     

Modulation of rat liver S-adenosylmethionine synthetase activity by glutathione.

Rat liver S-adenosylmethionine (AdoMet) synthetase appears as high-M(r) (tetramer) and low-M(r) (dimer) forms. Both are inhibited in the presence of GSSG at pH 8. The calculated Ki values are 2.14 and 4.03 mM for the high- and low-M(r) forms, respectively. No effect on enzyme activity was observed in the presence of GSH, but modulation of inhibition by GSSG can be obtained by addition of GSH. At a total glutathione concentration (GSH + GSSG) of 10 mM, a KOX of 1.74 was calculated for the high-M(r) form, whereas this constant was 2.85 for the low-M(r) AdoMet synthetase. No incorporation of [35S]GSSG was observed in either of the enzyme forms, and inhibition of enzyme activity was correlated with dissociation of both AdoMet synthetases to a monomer. The data obtained in the presence of GSSG seem to suggest that oxidation leads to the formation of an intrasubunit disulfide. The possible regulation of AdoMet synthetase activity by the GSH/GSSG ratio is discussed, as well as its in vivo significance.     

Characterization of a novel alpha-class anionic glutathione S-transferase isozyme from human liver

A novel, alpha-class glutathione S-transferase (GST) isozyme has been isolated from human liver using glutathione (GSH) affinity chromatography, DEAE-cellulose ion-exchange chromatography, and immunoaffinity chromatography. The isozyme is a dimer of approximately 25,000 Mr with blocked N termini. Structural, kinetic, and immunological properties of this enzyme indicate that it belongs to the alpha class of GSTs. Noticeable differences between the properties of this enzyme and the other alpha-class GSTs of human liver are its anionic nature (pI 5.0), GSH peroxidase activity toward hydrogen peroxide, and relatively higher GSH conjugating activities toward CDNB and epoxide substrates as compared to other alpha-class GSTs. Results of these studies indicate that anionic GST omega characterized previously (Y. C. Awasthi, D. D. Dao, and R. P. Saneto, 1980, Biochem. J. 191, 1-10) from human liver is a mixture of GST pi and a novel alpha-class GST. We have, therefore, reassigned the name GST omega to this new alpha-class anionic GST of human liver.     

Effects of trivalent antimony on human erythrocyte glutathione-S-transferases

Trivalent antimony (SB3+) in the form of potassium antimony tartrate was found to be an inhibitor of glutathione-S-transferases (GST) from human erythrocytes with a 50% inhibition concentration (IC50) of 0.05 mM. The inhibition was, however, incomplete with 15-20% of the GST activity remaining unaffected. In comparison, ethacrynic acid, a known inhibitor of GST, was tenfold more potent and affected close to 100% inhibition. Pentavalent antimony (SB5+) in the form of sodium stibogluconate had no effect on GST. Group V metalloids such as arsenite was slightly inhibitory, and arsenate was noninhibitory. When compared with five heavy metals, the inhibitory potency followed the order of SB3+ > Hg2+, Cu2+ > Cd 2+ > Cr3+ > Fe2+ x SB3+ inhibition of GST was competitive against the substrate 1-chloro-2,4-dinitrobenzene (CDNB) with an apparent Ki of 0.018 mM. Increasing the glutathione (GSH) concentration, however, produced a biphasic response: at concentrations below 1 mM, GSH was noncompetitive against SB3+, but at 1 mM and higher it was apparently competitive. A concurrent study of interactions between GSH, CDNB, and SB3+ showed that there was a significant nonenzymatic conjugation of CDNB at high GSH concentrations, which was suppressed by SB3+. The presence of albumin (500 mg/dL), or up to 5 mM N-acetylcysteine, cysteine, or ethylenediamine tetraacetic acid (EDTA) did not protect GST from the inhibitory effect of SB3+. The ability of erythrocyte GST to conjugate CDNB, which was measured directly by the formation of dinitrophenyl-glutathione (DNP-glutathione), was reduced by approximately 20 and 33%, respectively, in the presence of 2 and 10 mM SB3+, and nearly abolished with the addition of 0.2 mM ethacrynic acid. Based on these inhibition characteristics and the preferential accumulation of SB3+ in mammalian erythrocytes, it may be deduced that in the case of high antimonial intake, for example, during therapeutic treatment of Leishmaniasis, SB3+ levels in erythrocytes may be high enough to depress GST activity, which might compromise the ability of erythrocytes to detoxify electrophilic xenotbiotics.     

Allosteric and non-allosteric forms of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase: differential inhibition of activity by adenosine 2'-monophospho-5'-diphosphoribose

Adenosine 2'-monophospho-5'-diphosphoribose (P-ADP-Rib) is a structural analog of NADPH which was reported to competitively inhibit (Kiapp = 21.7 microM) solubilized rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Tanazawa, K., and A. Endo. 1979. Eur. J. Biochem. 98: 195-201). However, microsomal HMG-CoA reductase, which at low thiol concentrations exhibits allosteric properties, is only poorly inhibited by P-ADP-Rib (Kiapp = 550 microM at 4.5 mM GSH). Gradual shift of the microsomal reductase towards a non-allosteric form by increasing glutathione (GSH) concentrations resulted in a higher inhibition by P-ADP-Rib. Under these conditions, Ki values for P-ADP-Rib were 165 microM and 53 microM at 9 mM and 27 mM GSH, respectively. The largest change in the degree of inhibition by P-ADP-Rib was observed within the 10 mM range of GSH. By contrast, freeze-thaw solubilized HMG-CoA reductase, which does not display allosteric properties, is readily inhibited by P-ADP-Rib, even when assayed at a low concentration of GSH (Kiapp = 50 microM at 4.5 mM GSH). Assaying the solubilized reductase in the presence of increased thiol concentration results in a minor decrease in the apparent Ki for P-ADP-Rib (22 microM at 27 mM GSH). Microsomal HMG-CoA reductase is allosterically activated by various nucleotides. When activated by NADH, the enzyme is effectively inhibited by P-ADP-Rib even at a 4.5-mM GSH concentration (Kiapp = 175 microM in the presence of 300 microM NADH).(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of tyrosine residues in mitochondrial aspartate aminotransferase from beef kidney.

Mitochondrial aspartate aminotransferase from beef kidney is 50% inhibited after 2 hr treatment with 2.5 mM tetranitromethane at pH 8. Two tyrosine residues per enzyme protomer (46,000 daltons) are modified by the reagent either in the holoenzyme or in the apoenzyme. In both cases the five SH groups titratable with p-mercuribenzoate are not modified by the reagent. However, with a tetranitromethane concentration higher than 2.5 mM and 10 mM mercaptoethanol, an additional tyrosine residue is nitrated in both holo- and apoenzymes. These results are not affected by the presence in the incubation mixture of the substrates alpha-ketoglutarate and glutamate both at ten times their Km values. Mercaptoethanol does not impair the recombination of native or nitrated apoenzyme with the coenzyme and does not reduce the coenzyme moiety of native or nitrated holoenzyme, but promotes a conformational change in the nitrated holoenzyme which causes inactivation. Hydrosulfite promotes the reduction of the coenzyme moiety of native and nitro holoenzyme resulting in their inactivation, largely in the nitrated form. The recombination of the coenzyme with native or nitrated apoenzyme is not influenced by hydrosulfite.     

Crystal structures of the yeast prion Ure2p functional region in complex with glutathione and related compounds.

The [URE3] phenotype in yeast Saccharomyces cerevisiae is due to an altered prion form of Ure2p, a protein involved in nitrogen catabolism. To understand possible conformational changes at the origin of prion propagation, we previously solved the crystal structure of the Ure2p functional region [Bousset et al. (2001) Structure 9, 39-46]. We showed the protein to have a fold similar to that of the beta class of glutathione S-transferases (GSTs). Here we report crystal structures of the Ure2p functional region (extending from residues 95-354) in complex with glutathione (GSH), the substrate of all GSTs, and two widely used GST inhibitors, namely, S-hexylglutathione and S-p-nitrobenzylglutathione. In a manner similar to what is observed in many GSTs, ligand binding is not accompanied by a significant change in the conformation of the protein. We identify one GSH and one hydrophobic electrophile binding site per monomer as observed in all other GSTs. The sulfur group of GSH, that conjugates electrophiles, is located near the amide group of Asn124, allowing a hydrogen bond to be formed. Biochemical data indicate that GSH binds to Ure2p with high affinity. Its binding affects Ure2p oligomerization but has no effect on the assembly of the protein into amyloid fibrils. Despite results indicating that Ure2p lacks GST activity, we propose that Ure2p is a member of the GST superfamily that may describe a novel GST class. Our data bring new insights into the function of the Ure2p active region.     

Single-step purification and h.p.l.c. analysis of glutathione transferase 8-8 in rat tissues.

GSSG selectively elutes two GSH transferases from a mixture of rat GSH transferases bound to a GSH-agarose affinity matrix. One is a form of GSH transferase 1-1 and the other is shown to be GSH transferase 8-8. By using tissues that lack this form of GSH transferase 1-1 (e.g. lung), GSH transferase 8-8 may thus be purified from cytosol in a single step. Quantitative analysis of the tissue distribution of GSH transferase 8-8 was obtained by h.p.l.c.     

Quantification of human hepatic glutathione S-transferases.

Human hepatic glutathione S-transferase (GST) subunits were characterized and quantified with the aid of a recently developed h.p.l.c. method. In 20 hepatic tissue specimens the absolute amounts of the basic Class Alpha subunits B1 and B2, the near-neutral Class Mu subunits mu and psi and the acidic subunit pi were determined. The average total amount of GST was 37 micrograms/mg of cytosolic protein, with the Class Alpha GST being the predominant class (84% of total GSTs), and pi as the sole representative of the Class Pi GSTs present in the lowest concentration (4% of total GSTs). Large interindividual differences were observed for all subunits, with variations up to 27-fold, depending on the subunit. For the Class Alpha GST-subunits B1 and B2, a biphasic ratio was observed. The genetic polymorphism of the subunits mu and psi was confirmed by h.p.l.c. analysis, and correlated with the enzymic glutathione conjugation of trans-stilbene oxide and with Western blotting of cytosols, using a monoclonal anti-(Class Mu GST) antibody. Of the 20 livers examined, ten contained only mu, whereas the occurrence of psi alone, and the combination of mu and psi, were found in only one liver each.     

Characterization of a cold-adapted glutathione synthetase from the psychrophile Pseudoalteromonas haloplanktis

Glutathione (GSH) biosynthesis occurs through two ATP-dependent reactions, usually involving distinct enzymes; in the second step of this process, catalysed by glutathione synthetase (GshB), GSH is formed from γ-glutamylcysteine and glycine. A recombinant form of GshB from the cold-adapted source Pseudoalteromonas haloplanktis (rPhGshB) was purified and characterised. The enzyme formed a disulfide adduct with β-mercaptoethanol, when purified in the presence of this reducing agent. The homotetrameric form of rPhGshB observed at high protein concentration disassembled into two homodimers at low concentration. A new method for directly determining the rPhGshB activity was developed, based on [γ-(32)P]ATP hydrolysis coupled to the GSH synthesis. The ATPase activity required the presence of both γ-glutamylcysteine and glycine and its optimum was reached in the 7.4-8.6 pH range; a divalent cation was absolutely required for the activity, whereas monovalent cations were dispensable. rPhGshB was active at low temperatures and had a similar affinity for ATP (K(m) 0.26 mM) and γ-glutamylcysteine (K(m) 0.25 mM); a lower affinity was measured for glycine (K(m) 0.75 mM). The oxidised form of glutathione (GSSG) acted as an irreversible inhibitor of rPhGshB (K(i) 10.7 mM) and formed disulfide adducts with the enzyme. rPhGshB displayed a great temperature-dependent increase in its activity with an unusually high value of energy of activation (75 kJ mol(-1)) for a psychrophilic enzyme. The enzyme was moderately thermostable, its half inactivation temperature being 50.5 °C after 10 min exposure. The energy of activation of the heat inactivation process was 208 kJ mol(-1). To our knowledge, this is the first contribution to the characterization of a GshB from cold-adapted sources.     

Effect of hypoxic cell radiosensitizers on glutathione level and related enzyme activities in isolated rat hepatocytes

A comparative study of the effect of misonidazole and novel radiosensitizers on glutathione (GSH) levels and related enzyme activities in isolated rat hepatocytes was performed. Incubation of hepatocytes with 5 mM radiosensitizers led to a decrease in the intracellular GSH level. The most pronounced decrease in cellular GSH was evoked by 2,4-dinitroimidazole-1-ethanol (DNIE); after incubation for only 15 min, GSH was hardly detected. DNIE-mediated GSH loss was dependent upon its concentration. DNIE reacted with GSH nonenzymatically as well as with diethylmaleate, while misonidazole and 1-methyl-2-methyl-sulfinyl-5-methoxycarbonylimidazole (KIH-3) did not. Addition of partially purified glutathione S-transferase (GST) did not enhance DNIE-mediated GSH loss in a cell-free system. DNIE inhibited glutathione peroxidase (GSH-Px), GST, and glutathione reductase (GSSG-R) activities in hepatocytes, while misonidazole and KIH-3 did not. GSH-Px activity assayed with H2O2 as substrate was the most inhibited. Inhibition of GSH-Px activity assayed with cumene hydroperoxide as substrate and GST was less than that of GSH-Px assayed with H2O2 as substrate. GSSG-R activity was decreased by DNIE, but not significantly. Incubation of purified GSH-Px with DNIE resulted in a little change in the activity when assayed with H2O2 as substrate.     

Stimulation of thiamine diphosphatase activity by ascorbic acid in rat brain microsomes.

The effect of ascorbic acid on microsomal thiamine diphosphatase activity in rat brain was examined. Ascorbic acid at 0.02--0.1 mM increased the thiamine diphosphatase activity by 20--600% and produced a significant amount of lipid peroxide, which was measured with thiobarbiturate under the same conditions as the enzyme. A lag period of about 10 min was observed in the process of stimulation of enzyme activity by ascorbic acid. The stimulation of enzyme activity and the lipid peroxidation induced by ascorbic acid were blocked by metal-binding compounds (EDTA, alpha,alpha'-dipyridyl, o-phenanthroline) and an antioxidant (N,N'-diphenyl p-phenylenediamine). GSH significantly enhanced the stimulation of enzyme activity and formation of lipid peroxide by 0.02--0.05 mM ascorbic acid. The effect of GSH was due in part to maintenance of the concentration of ascorbic acid in the medium, since GSH could convert dehydroascorbic acid, an oxidized form of ascorbic acid, to ascorbic acid.     

Modulation of glyceraldehyde-3-phosphate dehydrogenase inAnacystis nidulans by glutathione

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13; GAPDH) from the cyanobacteriumAnacystis nidulans was activated up to five-fold by reduced glutathione (GSH) in the physiological concentration range (0.1–2 mM GSH). Non-physiological reductants, like dithiothreitol (DTT) and -mercaptoethanol, also activated the enzyme. Oxidized glutathione (GSSG) had no effect on the cyanobacterial GAPDH but treatment with H₂O₂ led to a rapid, reversible deactivation of both untreated and GSH-treated enzyme preparations. GSH reversed the inhibition induced by H₂O₂. An oligomeric form of the enzyme (apparentM _r440,000) was dissociated by GSH into a lower-M _r, more active enzyme form (M _r200,000). The enzyme was shown to obey regular Michaelis-Menten kinetics. The activation of GAPDH by GSH was associated with a decrease inK _m and an increase inV _max values of the enzyme for 3-phosphoglycerate. GSH had virtually no effect on a GAPDH preparation isolated from corn chloroplasts and studied for comparison.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase - GSH reduced glutathione - GSSG oxidized glutathione - DTT dithiothreitol     

Purification and characterization of glutathione transferases with an activity toward nitroglycerin from human aorta and heart. Multiplicity of the human class Mu forms

Although recent studies suggest involvement of glutathione transferase (GST) of blood vessels in vasodilation by nitroglycerin, GST forms in blood vessels remain to be studied. In this study, three GST forms (pI values 8.3, 6.6, and 4.8) were purified from human aorta and four (pI values 6.0, 5.6, 5.3, and 4.6) from the heart by affinity chromatography followed by chromatofocusing. The major form of both aorta (pI 4.8) and heart (pI 4.6) was identified as GST-pi, and the other five forms were immunologically related to GST-mu, suggesting that the five belong to the Mu class. Among nine human GST forms, including three in the Alpha class purified from the liver, GST-mu, aorta pI 8.3 form, and GST-I (a form of the Alpha class, corresponding to GST-epsilon (B1B1)) showed high activities toward nitroglycerin, 1.08, 0.85, and 0.78 units/mg protein, respectively. GST-pi did not exhibit the activity. The Km values of the aorta form (pI 8.3) for glutathione (GSH) and nitroglycerin were calculated as 0.12 and 1.1 mM, respectively. The Km values of GST-mu and GST-I for GSH were 0.29 and 0.09 mM, and those for nitroglycerin were 2.5 and 0.3 mM, respectively. The activity of the pI 8.3 form as well as GST-mu toward nitroglycerin was inhibited by bromosulfophthalein, which is known to inhibit the relaxation of rabbit aorta induced by nitroglycerin, at the lower concentration (IC50, 2 microM) than was GST-I (IC50, 32 microM). Two-dimensional gel electrophoresis and N-terminal amino acid sequence analysis revealed that five forms in the Mu class are homo- or heterodimers of five different subunits named M1 (pI 7.0/Mr 27,000), M2 (6.6/27,000), M3 (6.0/27,000), N1 (6.5/26,500), and N2 (5.9/26,500). The subunit structures of the five forms are as follows: pI 8.3 form, M1M2; 6.6 form, M2N1; 6.0 form, M3M3; 5.6 form, M3N2; and 5.3 form, N2N2. M3 and N2 seem to correspond to the subunits of GST-mu, and -4 (Board, P. G., Suzuki, T., and Shaw, D. C. (1988) Biochim. Biophys. Acta 953, 214-217), respectively. These subunits except N1 are different from each other at two or three positions in the first 20 residues of N-terminal amino acid sequence. These results indicate the presence of five different subunits in the human Mu class and also suggest that GST-M1M2 and -M2N1 found in the aorta are involved in the expression of the pharmacologic effect of nitroglycerin.     

溴化1-己基-3-甲基咪唑对斜生栅藻谷胱甘肽及其代谢酶的影响

研究了浓度为0、1、5、10、15、20 mg/L的新兴离子液体溴化1-己基-3-甲基咪唑([C6mim]Br)在24h、48h、72h和96h对斜生栅藻还原型谷胱甘肽（GSH）及其代谢酶-谷胱甘肽过氧化物酶（GPX）、谷胱甘肽转硫酶（GST）和谷胱甘肽还原酶（GR）的影响。结果表明：GSH含量在24h、48h和72h时,在最低处理浓度下不变,其他处理浓度下随胁迫浓度增加而降低,96h时则与对照无差异或较小;GPX和GST的活性在72h之前明显升高（最高浓度组的GST活性有波动）,96h时均降低至对照水平;GR活性在24h时,[C6mim]Br=1 mg/L时升高,之后降低,在48h增高至对照水平,72h时,[C6mim]Br≥10 mg/L的处理组高于对照水平,96h时,除最低处理组外,均降至对照水平以下。GR是GSH系统中的限速酶,GST则是该系统中活性和灵敏性最高的酶,可作为[C6mim]Br胁迫时的敏感的生物标志物。1 mg/L的[C6mim]Br可引起藻细胞的氧化胁迫,具有环境毒性。