Molecular characterization of corn glutathione S-transferase isozymes involved in herbicide detoxication

Corn ( Zea mays L.) glutathione S-transferases (EC 2.5.1.18) have attracted interest, in part, due to their involvement in the metabolism of several herbicides, including atrazine and alachlor. Three corn, glutathione S-transferases have been purified, and cDNA clones have been isolated and sequenced for two of these, GST I and GST III. In addition to showing some amino acid sequence similarity to each other, the two sequenced corn glutathione S-transferases also show some similarity to rat and human enzymes. The corn glutathione S-transferases responsible for atrazine tolerance have not yet been purified or cloned, but purification attempts indicate that corn has two glutathione S-transferases with activity towards atrazine. While many glutathione S-transferases from various organisms have been detected by using 1-chloro-2,4-dinitrobenzene as a substrate, the atrazine-specific glutathione S-transferases have very little or no activity with 1-chloro-2,4-dinitrobenzene. This shows the importance of assaying with a variety of substrates when characterizing glutathione S-transferases.     

The glutathione S-transferase activity in the kidney of rainbow trout (Salmo gairdneri)

Trout kidney contains 2.3 mmol GSH/kg. The cytosolic glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate is 0.35 mumol/min/mg protein. There is no detectable activity with 1,2-epoxy-3-(p-nitrophenoxy)propane, ethacrynic acid, p-nitrobenzyl chloride or p-nitrophenyl acetate. A variable proportion of the activity does not bind to a glutathione-affinity matrix. Its Km values for GSH and 1-chloro-2,4-dinitrobenzene are 3.0 and 5.1 mM, respectively. The rest of the activity is eluted from the affinity matrix as one main and two minor peaks. The main peak has Km values for GSH and 1-chloro-2,4-dinitrobenzene of 0.4 and 4.5 mM, respectively. Its subunit Mr is 22,900. The activity in the main peak is inhibited progressively by 1-chloro-2,4-dinitrobenzene with a rate constant of 0.11/min.     

Purification and some properties of glutathione S-transferase from Escherichia coli B.

Glutathione S-transferase was purified approximately 2,300-fold from cell extracts of Escherichia coli B with a 7.5% activity yield. The molecular weight of the enzyme was 45,000, and the enzyme appeared to consist of two homogeneous subunits. The enzyme was almost specific to 1-chloro-2,4-dinitrobenzene (Km, 1.43 mM) and glutathione (Km, 0.33 mM). The optimal pH and optimal temperature for activity were 7.0 and 50 degrees C, respectively, and the enzyme was stable from pH 5 to 11. The activity of the enzyme for 1-chloro-2,4-dinitrobenzene (3,2 mumol/min per mg of protein) was significantly lower than those of the enzymes from mammals, plants, and fungi.     

Hepatic and extra-hepatic glutathione-S-transferase activity in wild pigeons (Columba livia)

Glutathione-S-transferase (EC 2.5.1.18) activity was assayed in hepatic and extra-hepatic tissues of pigeons using l-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates. Gluthathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene in pigeon was in the order: kidney > liver > testes > brain > lung> heart. The enzyme activity with 1-chloro-2,4-dinitrobenzene as substrate was 40–44 times higher in pigeon liver and kidney than that observed with 1,2-dichloro-4-dinitrobenzene as substrate.K _m values of hepatic and renal glutathione transferase with l-chloro-2,4-dinitrobenzene as substrate were 2.5 and 3 mM respectively. Double reciprocal plots with varying reduced gluthathione concentrations resulted in biphasic curves with twoK _m values (liver 0.31 mM and 4mM; kidney 0.36 mM and 1.3 mM). The enzyme activity was inhibited by oxidized gluthathione in a dose-dependent pattern. 3-Methylcholanthrene elicited about 50% induction of hepatic glutathione transferase activity whereas phénobarbital was ineffective.     

Glutathione S-transferase composition of rat erythrocytes

With 1-chloro-2,4-dinitrobenzene as the electrophilic substrate, the specific activity of glutathione S-transferase in rat haemolysates was found to range from 0.002 to 0.013 mumol/min/mg haemoglobin at 30 degrees C. To establish the glutathione S-transferase composition, chromatofocusing was used which indicated the presence of a single soluble isoenzyme with an apparent pI of 6.1. A molecular weight of 48,000 was determined for the enzyme by gel filtration. The transferase enzyme in intact erythrocytes is shown to catalyze the formation of S-(2,4-dinitrophenyl)-glutathione from 1-chloro-2,4-dinitrobenzene and endogenous glutathione. Efflux of this conjugate from erythrocytes proceeded at a rate of 13 nmol/min/ml at 37 degrees C.     

The glutathione S-transferase activity in the pyloric caeca of rainbow trout, Salmo gairdneri

Pyloric caeca of trout contain 1.9 mmol GSH/kg tissue. Cytosolic glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate is 0.06 mmol/min/g protein. Cholate (3.3 mM) inhibits cytosolic transferase activity by 55% at pH 6.6 and by 4% at pH 7.4. The transferases do not bind 8-anilino-1-naphthalene sulphonate at pH 7.4. The cytosolic transferases are inactivated progressively by 1-chloro-2,4-dinitrobenzene, 50% of their activity being lost in 5.0 min. A minority of the activity does not bind to a glutathione-affinity matrix. At pH 6.6 its apparent Michaelis constants for GSH and 1-chloro-2,4-dinitrobenzene are 0.88 and 9.1 mM respectively. The rest of the activity is eluted from the affinity matrix as a single peak. Its apparent Michaelis constants for GSH and 1-chloro-2,4-dinitrobenzene are 0.33 and 2.9 mM respectively. Its subunit Mr is 22.4 kDa.     

Significance of glutathione S-conjugate for glutathione metabolism in human erythrocytes

The significance of glutathione S-conjugate in the regulation of glutathione synthesis was studied using human erythrocyte gamma-glutamylcysteine synthetase. Feedback inhibition of the enzyme by reduced glutathione was released by the addition of the glutathione S-conjugate (S-2,4-dinitrophenyl glutathione). A half-maximal effect of glutathione S-conjugate on gamma-glutamylcysteine synthetase activity was obtained at approximately 1 microM; 50 microM glutathione S-conjugate in the presence of 10 mM glutathione actually increased the enzyme activity twofold above uninhibited levels. Glutathione S-conjugate had no effect on the enzyme activity in the absence of glutathione. When erythrocytes were exposed to the electrophile 1-chloro-2,4-dinitrobenzene, which forms a glutathione S-conjugate by the catalytic reaction of glutathione S-transferase, the level of glutathione synthesis increased. These data suggest that glutathione S-conjugate plays a role in stimulating the synthesis of glutathione.     

Purification and characterization of a new form of glutathione S-transferase from human erythrocytes

Presence of a new form of glutathione S-transferase has been demonstrated in human erythrocytes. using two different affinity ligands this enzyme has been separated from the previously characterized glutathione S-transferases ?. The new enzyme is highly basic with a pI of > 10. The new enzyme which represents less than 5 percent of glutathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene as substrate and about 10 percent of total glutathione S-transferase protein of erythrocytes has different amino acid composition, substrate specificities, and immunological characteristics from those of the major erythrocyte glutathione S-transferase ?. Immunological properties of the new enzyme indicate that this form may be different from other glutathione S-transferases of human tissues.     

Purification and cloning of a Delta class glutathione S-transferase displaying high peroxidase activity isolated from the German cockroach Blattella germanica

A highly active glutathione S-transferase was purified from adult German cockroaches, Blattella germanica. The purified enzyme appeared as a single band of 24 kDa by SDS/PAGE, and had a different electrophoretic mobility than, a previously identified Sigma class glutathione S-transferase (Bla g 5). Kinetic study of 1-chloro-2,4-dinitrobenzene conjugation revealed a high catalytic rate but common substrate-binding and cosubstrate-binding affinities, with V(max), k(cat), K(m) for 1-chloro-2,4-dinitrobenzene and K(m) for glutathione estimated to be 664 micromol x mg(-1) x min(-1), 545 s(-1), 0.33 mm and 0.76 mm, respectively. Interestingly, this enzyme possessed the highest activity for cumene hydroperoxide among insect glutathione S-transferases reported to date. Along with the ability to metabolize 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane and 4-hydroxynonenal, this glutathione S-transferase may play a role in defense against insecticides as well as oxidative stress. On the basis of the amino acid sequences obtained from Edman degradation and MS analyses, a 987-nucleotide cDNA clone encoding a glutathione S-transferase (BggstD1) was isolated. The longest ORF encoded a 24 614 Da protein consisting of 216 amino acid residues. The sequence had close similarities ( approximately 45-60%) to that of Delta class glutathione S-transferases, but had only 14% identity to Bla g 5. The putative amino acid sequence contained matching peptide fragments of the purified glutathione S-transferase. ELISA showed that BgGSTD1 bound to serum IgE obtained from patients with cockroach allergy, indicating that the protein may be a cockroach allergen.     

Developmental aspects of glutathione S-transferase B (ligandin) in rat liver.

The postnatal development in male Sprague-Dawley rats of hepatic glutathione S-transferase B (ligandin) in relation to the other glutathione S-transferases is described. The concentration of glutathione S-transferase B in 1-day-old male rats is about one-fifth of that in adult animals. The enzyme reaches adult concentrations 4-5 weeks later. When assessed by substrate specificity or immunologically, the proportion of transferase B relative to the other glutathione S-transferases is high during the first week after birth. At this age, 67.5% of the transferase activity towards 1-chloro-2,4-dinitrobenzene is immunoprecipitable by anti-(transferase B), compared with about 50% in adults and older pups. Between the second and the fifth postnatal week, the fraction of transferase B increases in parallel fashion with the other transferases in hepatic cytosol. Neither L-thyroxine nor cortisol induce a precocious increase in glutathione S-transferase activity. Phenobarbital did induce transferase activity towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene in both pups and adults. The extent of induction by phenobarbital was a function of basal activity during development such that the percentage stimulation remained constant from 5 days postnatally to adulthood.     

Effect of microsomal enzyme inducing agents on hepatic biotransformation in cotton rats (Sigmodon hispidus): comparison to that in Sprague-Dawley rats.

1. Activities of several biotransformation enzymes were determined in male and female Sigmodon hispidus. Benzphetamine N-demethylase and glutathione S-transferases toward 1-chloro-2,4-dinitrobenzene and sulfobromophthalein were higher in male Sigmodon hispidus than the female animals. 2. The study also determined the effect of microsomal enzyme inducing agents on hepatic biotransformation in male Sigmodon hispidus. 3. Cytochrome P-450 concentration was similar in cotton and Sprague-Dawley rats, and was increased after phenobarbital, pregnenolone-16 alpha-carbonitrile, or 3-methylcholanthrene treatment. 4. Benzphetamine N-demethylase was 4-fold higher in Sigmodon hispidus and was induced by 75-100% after phenobarbital. 5. UDP-Glucuronosyltransferase toward estrone, 1-naphthol, diethylstilbestrol and testosterone was 2- to 4-fold higher in cotton rats and was not altered by treatment with the inducing agents. 6. Conjugation of 1-chloro-2,4-dinitrobenzene, ethacrynic acid and sulfobromophthalein with glutathione was similar in both rodent species and was not inducible. 7. Sulfation of 2-naphthol was 15-30% of that in Sprague-Dawley rats and was not increased by inducer administration.     

Bioconversion of isosorbide dinitrate into isosorbide mononitrate by the protozoan Tetrahymena thermophila: relationship to glutathione transferase levels

Summary Growing cells of Tetrahymena thermophila (T. t.) metabolized, after 72 h, 80% of isosorbide dinitrate (ISDN) to isosorbide 5-mononitrate (5-ISMN) and isosorbide 2-mononitrate (2-ISMN) in a ratio 5-ISMN/2-ISMN=2.6 as evaluated by HPLC. The level of glutathione S-transferases (GSH-ST) determined by following the reactions with of 1-chloro-2,4-dinitrobenzene (CDNB), o-dinitrobenzene (o-DNB) or ISDN, showed the inductive effect of ISDN (0.5 mg/ml) on the level of this enzyme. The enzymatic activity, evaluated at 72 h, showed a twofold increase compared with the control. The GSH-ST activity correlated well with the rate of ISDN bioconversion.     

Trialkyl phosphorothioates and glutathione S-transferases

Using a rat liver cytosol source of enzyme trialkyl phosphorothioates have been shown to be substrates of glutathione S-transferases. Using OSS-trimethyl phosphorodithioate (OSS-Me(O] and OOS-trimethyl phosphorothioate (OOS-Me(O] the methyl transferred to the sulphydryl of glutathione is that attached to phosphorus via an oxygen atom. Fractionation of liver cytosol has shown that although the bulk activity is due to the three isozymes (1-1; 3-4; 1.2), OSS-Me(O) is a general substrate for glutathione S-transferases. The specific activity is low compared with the substrates 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene.     

Optical biosensor consisting of glutathione-S-transferase for detection of captan

The optical biosensor consisting of a glutathione-S-transferase (GST)-immobilized gel film was developed to detect captan in contaminated water. The sensing scheme was based on the decrease of yellow product, s-(2,4-dinitrobenzene) glutathione, produced from substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), due to the inhibition of GST reaction by captan. Absorbance of the product as the output of enzyme reaction was detected and the light was guided through the optical fibers. The enzyme reactor of the sensor system was fabricated by the gel entrapment technique for the immobilized GST film. The immobilized GST had the maximum activity at pH 6.5. The optimal concentrations of substrates were determined with 1 mM for both of CDNB and GSH. The optimum concentration of enzyme was also determined with 100 μg/ml. The activity of immobilized enzyme was fairly sustained during 30 days. The proposed biosensor could successfully detect the captan up to 2 ppm and the response time to steady signal was about 15 min.     

Inhibition of purified glutathione S-transferases by indomethacin

Soluble rat liver glutathione S-transferases have been purified and a previously undescribed peak was observed. This peak contained glutathione S-transferase activity which was extensively inhibited by indomethacin. Glutathione conjugation of 1-chloro-2,4-dinitrobenzene by this isozyme, designated glutathione S-transferase VII, was inhibited 44 and 68% at indomethacin concentrations of 0.20 and 1.00 microM, respectively. The other six basic glutathione S-transferase isozymes were relatively unaffected by low concentrations of indomethacin. The pharmacological significance of this inhibition by indomethacin is largely dependent on the role of the glutathione S-transferase VII in leukotriene synthesis.     

Inhibition of glutathione transferase pi from human placenta by 1-chloro-2,4-dinitrobenzene occurs because of covalent reaction with cysteine 47.

Human placenta glutathione transferase pi is irreversibly inhibited when incubated with 1-chloro-2,4-dinitrobenzene (CDNB) in the absence of the cosubstrate glutathione. The enzyme is protected against CDNB inactivation by the presence of S-methylglutathione and glutathione. The kinetics of inactivation is pseudo-first-order with k(obs) = 0.04 min-1 when 44 microM enzyme is incubated in presence of 1 mM CDNB at pH 6.5. The inhibition is saturable with respect to the CDNB concentration and the enzyme-CDNB complex shows a K(i) = 2.7 mM. Concomitant to the inhibition process is formation of an absorption band at 340 nm. After trypsin digestion and HPLC analysis, the CDNB-reacted enzyme gives a single peptide absorbing at 340 nm. Automated Edman degradation of this peptide indicates cysteine 47 to be the residue alkylated by CDNB.     

Purification and characterization of a novel glutathione S-transferase from Atactodea striata

A novel GST isoenzyme was purified from hepatopancreas cytosol of Atactodea striata with a combination of affinity chromatography and reverse-phase HPLC. The molecular weight of the enzyme was determined to be 24 kDa by SDS-PAGE electrophoresis and 48 kDa by gel chromatography, in combination with GST information from literature revealed that the native enzyme was homodimeric with a subunit of M(r) 24 kDa. The purified enzyme, exhibited high activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Kinetic analysis with respect to CDNB as substrate revealed a K(m) of 0.43 mM and V(max) of 0.24 micromol/min/mg and a specific activity of 108.9 micromol/min/mg. The isoelectric point of the enzyme was 5.5 by isoelectric focusing and its optimum temperature was 38 degrees C and the enzyme had a maximum activity at approximately pH 8.0. The amino acid composition was also determined for the purified enzyme.     

MALDI mass sequencing and characterization of filarialglutathione-S-transferase

Glutathione-S-transferase has been detected in the somatic extract and excretory-secretory products of different life stages of Setaria cervi, a bovine filarial parasite. The enzyme was subjected to MALDI-TOF followed by mass spectrometry and the nearest match found was Pleuronectes platessa GST. Molecular mass of the purified enzyme was approximately 26 kDa as determined by SDS-PAGE and MALDI-TOF. Setaria cervi GST exhibited high activity towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene and glutathione as substrate revealed a K(m) of 2.22 mM and 0.61 mM, respectively. The activity was inhibited significantly by Cibacron blue and alpha-tocopherol.     

Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical Mr of 24,000 whereas a predominant subunit with Mr of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.     

Biochemical and immunological characterization of rat spleen prostaglandin D synthetase

Rat spleen prostaglandin D synthetase (Christ-Hazelhof, E., and Nugteren, D. H. (1979) Biochim. Biophys. Acta 572, 43-51) is very similar to rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi O. (1985) J. Biol. Chem. 260, 12410-12415) as judged by their pI (4.7-5.2), Mr (26,000-27,000), and self-inactivation during the isomerase reaction from prostaglandin H2 to prostaglandin D2. However, the amino acid compositions of these two enzymes were quite different. Furthermore, the spleen enzyme was associated with the glutathione S-transferase activity, differing from the brain enzyme. The synthetase and transferase activities of the spleen enzyme showed almost identical pH and glutathione dependencies, the optimum pH = 8.0 and Km for glutathione = 300 microM. The Km values for prostaglandin H2 and 1-chloro-2,4-dinitrobenzene (a substrate for the transferase) were about 200 microM and 5 mM, respectively. The synthetase activity was dose-dependently inhibited by 1-chloro-2,4-dinitrobenzene (IC50: approximately 5 mM) and more strongly by nonsubstrate ligands, such as bilirubin and indocyanine green (IC50: 150 and 2 microM, respectively). Both the synthetase and transferase activities of the purified enzyme dose-dependently decreased and showed identical immunotitration curves by incubation with antibody against this enzyme, but remained unchanged when treated with antibody against the brain enzyme. The antibody specific for the spleen enzyme absorbed almost all of the synthetase activity and about 10% of the transferase activity in the spleen, but not the transferase activity in the liver, heart, and testis. These results show that the two types of prostaglandin D synthetase are similar but different enzymes and that the spleen enzyme is a unique glutathione S-transferase differing from other isozymes and their subunits reported previously.