Soluble glutathione transferase isoenzymes in Daphnia magna straus and their interaction with 2,4-dichlorophenoxyacetic acid and 1,4-benzoquinone

The glutathione transferase (GST) activity in the cytosol of the water flea Daphnia magna Straus was partially purified by glutathione affinity chromatography. Chromatofocusing on the Polybuffer exchangers 94 and 118 separated the GST isoenzymes in one neutral and four cationic forms, and some minor fractions one of which was an anionic form. The major GST isoenzymes were partially characterized by different biochemical parameters. The water pollutants 2,4-dichlorophenoxyacetic acid and 1,4-benzoquinone inhibited the water flea GST isoenzymes, following the same kinetic inhibition patterns as for rat liver GST. It is concluded that water flea GST can play an important role in the detoxification of aquatic pollutants.     

Two glutathione S-transferase isoenzymes purified from Bulinus truncatus (Gastropoda: Planorbidae)

We purified and characterized two major glutathione S-transferase isoenzymes (GST2 and GST3) from snail Bulinus truncatus (Mollusca, Gastropoda, Planorbidae) tissue. The Km with respect to 1-chloro-2, 4-dinitrobenzene (CDNB) for both isoenzymes was increased as the pH decreased. Km of both isoenzymes with respect to glutathione (GSH) doubled when the pH was increased from 6.0 to 6.5. Acid inactivated GST2 and GST3 and the two enzymes were almost inactive at pH 3.5. However, they retain the full activity for at least 20 h when incubated at pH between 6.0 and 9.0. The optimum temperature was 45 degrees C for GST2 and 50 degrees C for GST3. The half lifetime at 50 degrees C was 70 min and 45 min for GST2 and GST3 isoenzymes, respectively. Addition of 5 mM GSH to the incubation buffer increased the half life of both isoenzymes more than fourfold. The activation energy for catalyzing the conjugation of CDNB was 1.826 and 3.435 kcal/mol for GST2 and GST3, respectively. I50 values for Cibacron blue, bromosulphophthalein, indocyanine green, hematin and ethacrynic acid were 0.76 microM, 47.9 microM, 7.59 microM, 0.03 microM and 0.79 microM for GST2, and 0.479 microM, 79.4 microM, 89.1 microM, 32.4 microM and 1.15 microM for GST3, respectively. Cibacron blue and indocyanine green were non-competitive inhibitors, while hematin was a mixed inhibitor. Bromosulphophthalein was found to be a competitive inhibitor for GST2 and a mixed inhibitor for GST3.     

Purification and partial characterization of the glutathione S-transferases in carp liver, and their interaction with 2,4-dichlorophenoxyacetic acid and 1,4-benzoquinone

The hepatic glutathione S-transferase (GST) activity in the cytosol of the freshwater fish carp (Cyprinus carpio) was enriched by glutathione affinity chromatography. The anionic (GST A1-A3) and cationic (GST C1-C3) isoenzymes were then separated in two chromatofocusing steps. SDS electrophoresis showed GST C1 to be a heterodimer with subunits of Mr 25,000 and 28,000, and all other isoenzymes to be homodimers with subunits of Mr 25,400. They were partially characterized by different biochemical parameters. The water pollutants 2,4-dichlorophenoxyacetic acid and 1,4-benzoquinone inhibited all carp GST isoenzymes, following the same kinetic inhibition patterns as for rat liver GST. It is concluded that hepatic carp GST can play an important role in the detoxication of aquatic pollutants.     

Purification and characterization of glutathione S-transferases of human kidney.

Several forms of glutathione S-transferase (GST) are present in human kidney, and the overall isoenzyme pattern of kidney differs significantly from those of other human tissues. All the three major classes of GST isoenzymes (alpha, mu and pi) are present in significant amounts in kidney, indicating that GST1, GST2 and GST3 gene loci are expressed in this tissue. More than one form of GST is present in each of these classes of enzymes, and individual variations are observed for these classes. The structural, immunological and functional properties of GST isoenzymes of three classes differ significantly from each other, whereas the isoenzymes belonging to the same class have similar properties. All the cationic GST isoenzymes of human kidney except for GST 9.1 are heterodimers of 26,500-Mr and 24,500-Mr subunits. GST 9.1 is a dimer of 24,500-Mr subunits. All the cationic isoenzymes of kidney GST cross-react with antibodies raised against a mixture of GST alpha, beta, gamma, delta and epsilon isoenzymes of liver. GST 6.6 and GST 5.5 of kidney are dimers of 26,500-Mr subunits and are immunologically similar to GST psi of liver. Unlike other human tissues, kidney has at least two isoenzymes (pI 4.7 and 4.9) associated with the GST3 locus. Both these isoenzymes are dimers of 22,500-Mr subunits and are immunologically similar to GST pi of placenta. Some of the isoenzymes of kidney do not correspond to known GST isoenzymes from other human tissues and may be specific to this tissue.     

Glutathione S-transferase isoenzymes from Streptomyces griseus

An inducible, cytosolic glutathione S-transferase (GST) was purified from Streptomyces griseus. GST isoenzymes with pI values of 6.8 and 7.9 used standard GST substrates including 1-chloro-2,4-dinitrobenzene. GST had subunit and native M(r)s of 24 and 48, respectively, and the N-terminal sequence SMILXYWDIIRGLPAH.     

The effects of selenium and copper deficiencies on glutathione S-transferase and glutathione peroxidase in rat liver.

Selenium (Se) deficiency in rats produced significant increases in the activity of hepatic glutathione S-transferase (GST) with 1-chloro-2,4-dinitrobenzene as substrate and in various GST isoenzymes when determined by radioimmunoassay. These changes is GST activity and concentration were associated with Se deficiency that was severe enough to provoke decreases of over 98% in hepatic Se-containing glutathione peroxidase activity (Se-GSHpx). However, decreases in hepatic Se-GSHpx of 60% induced by copper (Cu) deficiency had no effect on GST activity or concentration. Increased GST activity in Se deficiency has previously been postulated to be a compensatory response to loss of Se-GSHpx, since some GSTs have a non-Se-glutathione peroxidase (non-Se-GSHpx) activity. However, the GST isoenzymes determined in this study, GST Yb1Yb1, GST YcYc and GST YaYa, are known to have up to 30-fold differences in non-Se-GSHpx activity, but they were all significantly increased to a similar extent in the Se-deficient rats.     

Comparative studies on the isoenzymes of glutathione S-transferase of rat brain and other tissues

Six forms of glutathione S-transferase (GST) designated as GST 9.3, GST 7.5, GST 6.6, GST 6.1, GST 5.7 and GST 4.9 have been purified to homogeneity from rat brain. All GST isoenzymes of rat brain are apparent homodimers of one of the three type subunits, Ya, Yb, or Yc. More than 60% of total GST activity of rat brain GST activity is associated with the isoenzymes containing only the Yb type of subunits. In these respects brain GST isoenzymes differ from those of lung and liver. The Ya, Yb, and Yc type subunits of brain GST are immunologically similar to the corresponding subunits of liver and lung GST. The isoelectric points and kinetic properties of the Yb type subunit dimers in brain are strikingly different from those of the Yb type dimers present among liver GST isoenzymes indicating subtle differences between these subunits of brain and liver.     

Hepatic glutathione S-transferases in rainbow trout and their interaction with 2,4-dichlorophenoxyacetic acid and 1,4-benzoquinone

Glutathione S-transferase in the cytosol of rainbow trout liver was partially purified by affinity chromatography on a column with glutathione coupled to epoxy-activated Sepharose 6B, which retained 94% of the total activity. Chromatofocussing on a Polybuffer exchanger 118 column separated the glutathione S-transferase into six major cationic isoenzymes (K1-K6), and some minor fractions. SDS-polyacrylamide slab gel electrophoresis showed K1-K3 to be heterodimers with subunits of Mr 25,000 and 26,500, and K4-K6 to be homodimers with subunits of Mr 25,000. The glutathione S-transferase isoenzymes were partially characterized by different biochemical parameters. The hepatic rainbow trout glutathione S-transferases were inhibited by the organic water pollutants, 1,4-benzoquinone and 2,4-dichlorophenoxyacetic acid. The same kinetic inhibition patterns were observed with these inhibitors as for rat liver glutathione S-transferases. It is concluded that rainbow trout glutathione S-transferases can play a key role in the detoxication of organic micropollutants in the aquatic environment.     

Expression of human glutathione S-transferase 2 in Escherichia coli. Immunological comparison with the basic glutathione S-transferases isoenzymes from human liver.

A plasmid, termed pTacGST2, which contains the complete coding sequence of a GST2 (glutathione S-transferase 2) subunit and permits the expression of the protein in Escherichia coli was constructed. The expressed protein had the same subunit Mr as the enzyme from normal human liver and retained its catalytic function with both GST and glutathione peroxidase activity. Antiserum raised against the bacterially synthesized protein cross-reacted with all the basic GST isoenzymes in human liver. The electrophoretic mobility in agarose of the bacterially expressed isoenzyme suggested that its pI is identical with that of the cationic isoenzyme from human liver previously termed GST2 type 1. The available evidence suggests that the three common cationic isoenzymes found in human liver are the products of two very similar gene loci.     

Cationic glutathione S-transferase of human erythrocytes has unique kinetic characteristics among human glutathione S-transferases

The cationic glutathione S-transferase (GST sigma) of human erythrocytes is activated when incubated with 1 mM N-ethylmaleimide or other sulfhydryl blocking agents. Other GST isoenzymes of human tissues were inhibited by these reagents under similar conditions. At higher concentrations of NEM, GST sigma was also inhibited. Dithiothreitol, 2-mercaptoethanol, and sodium borohydride also caused several fold activation of GST sigma but noe of the other human GST isoenzymes were activated by these reagents.     

Oltipraz-induced decrease in the activity of cytosolic glutathione S-transferase in Schistosoma mansoni.

The activity of glutathione S-transferase (GST) decreased progressively in Schistosoma mansoni from mice treated with oltipraz (OPZ). However, the peroxidase activity of GST (selenium-independent) and selenium-dependent glutathione peroxidase was not affected by OPZ treatment. Purification and quantification of GST from worms after OPZ treatment indicated that the decrease in enzyme activity was greater than could be accounted for by the decrease in GST protein content. SDS-polyacrylamide gel electrophoresis followed by Western blot analysis with GST isoenzyme specific antisera revealed a slight decrease in the quantity of both 26 and 28 kDa GSTs. Fractionation of cytosolic GSTs from male S. mansoni by chromatofocusing resolved three major isoenzymes (SmI, II and III) and a minor form which eluted first from the column. SmI, II and III all had a molecular weight of about 28 kDa on SDS-polyacrylamide gel electrophoresis. However, on electrophoresis in the absence of SDS, the three GST forms exhibited different mobilities. The pattern of SmI, II and III was similar in untreated and OPZ-treated worms, but the activities of the isoenzymes from treated worms were lower. The results suggest that OPZ interacts with the GST isoenzymes SmI, II and III in a similar manner; thus, the effects are not isoenzyme specific. Taken together, these results suggest that OPZ and/or its metabolites interact directly with GST resulting in inhibition of activity and reduction in total enzyme protein. This mechanism may be important in the antischistosomal action of OPZ.     

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.     

Purification and properties of glutathione transferase from Issatchenkia orientalis.

Glutathione transferase (GST) (EC 2.5.1.18) was purified from a cell extract of Issatchenkia orientalis, and two GST isoenzymes were isolated. They had molecular weights of 37,500 and 40,000 and were designated GST Y-1 and GST Y-2, respectively. GST Y-1 and GST Y-2 gave single bands with molecular weights of 22,000 and 23,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. GST Y-1 and GST Y-2 were immunologically distinguished from each other. GST Y-1 showed specific activity 10.4-times and 6.0-times higher when 1-chloro-2,4-dinitrobenzene and o-dinitrobenzene were used as substrates, respectively, than GST Y-2. GST activity was not detected for either isoenzyme when other substrates such as bromosulfophthalein and trans-4-phenyl-3-buten-2-one were used. GST Y-1 and GST Y-2 had Km values of 0.51 and 0.75 mM for glutathione, respectively, and of 0.16 and 4.01 mM for 1-chloro-2,4-dinitrobenzene. GST Y-1 was significantly inhibited by Cibacron blue 3G-A, and GST Y-2 was significantly inhibited by bromosulfophthalein.     

Glutathione S-Transferase Isoenzymes from Streptomyces griseus

An inducible, cytosolic glutathione S-transferase (GST) was purified from Streptomyces griseus. GST isoenzymes with pI values of 6.8 and 7.9 used standard GST substrates including 1-chloro-2,4-dinitrobenzene. GST had subunit and native M_rs of 24 and 48, respectively, and the N-terminal sequence SMILXYWDIIRGLPAH.     

The development expression of alpha-, mu- and pi-class glutathione S-transferases in human liver

The developmental expression of the alpha, mu and pi class glutathione S-transferases has been defined in human liver using radioimmunoassay and immunohistochemistry. Expression of alpha and mu class isoenzymes increased significantly at birth, while that of the pi isoenzyme declined during the first trimester. Mu-class isoenzymes (GST1 1, GST1 2, GST1 2-1) were expressed in hepatocytes but not in other liver cell types.     

The major glutathione S-transferase in salmonid fish livers is homologous to the mammalian pi-class GST.

1. The hepatic glutathione S-transferase (GST) isoenzymes were isolated and characterized from salmon, sea trout and rainbow trout. 2. In all three species the predominant GST expressed comprised subunits of Mr 24,800. These subunits each co-migrated with the rat pi-class Yf polypeptide during SDS/polyacrylamide gel electrophoresis. 3. Western blotting experiments demonstrated immunochemical cross-reactivity between the major salmonid and the rat pi-class GSTs. 4. The salmon GST of subunit Mr 24,800 was digested with cyanogen bromide and the peptides, once purified by reverse-phase HPLC, were subjected to automated amino acid sequencing. 5. Over the region sequenced, the salmon GST possessed about 65% homology with the rat and human pi-class GST.     

Isoenzymes of glutathione S-transferase from the mosquito Anopheles dirus species B: the purification, partial characterization and interaction with various insecticides

Previously we have purified and characterized a major glutathione S-transferase (GST) activity, GST-4a, from the Thai mosquito Anopheles dirus B, a model mosquito for study of anopheline malaria vectors [Prapanthadara, L. Koottathep, S., Promtet, N., Hemingway, J. and Ketterman, A.J. (1996) Insect Biochem. Mol. Biol. 26:3, 277-285]. In this report we have purified an isoenzyme, GST-4c, which has the greatest DDT-dehydrochlorinase activity. Three additional isoenzymes, GST-4b, GST-5 and GST-6, were also partially purified and characterized for comparison. All of the Anopheles GST isoenzymes preferred 1-chloro-2,4-dinitrobenzene (CDNB) as an electrophilic substrate. In kinetic studies with CDNB as an electrophilic substrate, the V(max) of GST-4c was 24.38 micromole/min/mg which was seven-fold less than GST-4a. The two isoenzymes also possessed different K(m)s for CDNB and glutathione. Despite being only partially pure GST-4b had nearly a four-fold greater V(max) for CDNB than GST-4c. In contrast, GST-4c possessed the greatest DDT-dehydrochlorinase specific activity among the purified insect GST isoenzymes and no activity was detected for GST-5. Seven putative GST substrates used in this study were not utilized by An. dirus GSTs, although they were capable of inhibiting CDNB conjugating activity to different extents for the different isoenzymes. Bromosulfophthalein and ethacrynic acid were the most potent inhibitors. The inhibition studies demonstrate different degrees of interaction of the An. dirus isoenzymes with various insecticides. The GSTs were inhibited more readily by organochlorines and pyrethroids than by the phosphorothioates and carbamate. In a comparison between An. dirus and previous data from An. gambiae the two anopheline species possess a similar pattern of GST isoenzymes although the individual enzymes differ significantly at the functional level. The available data suggests there may be a minimum of three GST classes in anopheline insects.     

Differential activities of rat and human lung glutathione S-transferase isoenzymes towards benzo(a)pyrene epoxides

The isoenzymes of human and rat lung glutathione S-transferase (GST) differ among themselves in their activities towards the epoxides of benzo(a)pyrene (BP). The Ya' and Yc-type subunits of rat lung GST exhibit maximum activities towards BP-4,5-oxide and BP-7,8-oxide suggesting that these two subunits are preferentially involved in the detoxification of highly reactive epoxides and diol-epoxides of polycyclic aromatic hydrocarbons (PAH). The studies with human lung GST isoenzymes indicate that BP-4,5-oxide, and BP-7,8-oxide are preferred substrates for the cationic (pI 8.3) form of the enzyme. Identification of compounds which can selectively induce these isoenzymes of GST could prove useful as inhibitors of PAH induced neoplasia.     

Invited Commentary Potential Contribution of the Glutathione S-Transferase Supergene Family to Resistance to Oxidative Stress

The glutathione S-transferase (GST) supergene family comprises gene families that encode isoenzymes that are widely expressed in mammalian tissue cytosols and membranes. Both cytosolic (particularly the isoenzymes encoded by the alpha, mu and theta gene families) and microsomal GST catalyse the conjugation of reduced glutathione (GSH) with a wide variety of electrophiles which include known carcinogens as well as various compounds that are products of oxidative stress including oxidised DNA and lipid. Indeed, several lines of evidence suggest certain of these isoenzymes play a pivotal role in protecting cells from the consequences of such stress. An assessment of the importance of these GST in humans is presently difficult however, because the number of alpha and theta class genes is not known and, the catalytic preferences of even identified isoforms is not always clear.     

Invited Commentary Potential Contribution of the Glutathione S-Transferase Supergene Family to Resistance to Oxidative Stress

The glutathione S-transferase (GST) supergene family comprises gene families that encode isoenzymes that are widely expressed in mammalian tissue cytosols and membranes. Both cytosolic (particularly the isoenzymes encoded by the alpha, mu and theta gene families) and microsomal GST catalyse the conjugation of reduced glutathione (GSH) with a wide variety of electrophiles which include known carcinogens as well as various compounds that are products of oxidative stress including oxidised DNA and lipid. Indeed, several lines of evidence suggest certain of these isoenzymes play a pivotal role in protecting cells from the consequences of such stress. An assessment of the importance of these GST in humans is presently difficult however, because the number of alpha and theta class genes is not known and, the catalytic preferences of even identified isoforms is not always clear.