Acidic Glutathione S-transferase Research Articles

Antioxidant enzymes in the human iris: an immunogold study

AIMSTo determine the nature and extent of the antioxidant enzyme system in the human iris.METHODSThe fine structural distribution of five antioxidant enzymes (acidic, neutral, and basic glutathione S-transferase (GST), Cu/Zn...

Radioimmunoassay for plasma glutathione S-transferase-pi and its clinical application in gastrointestinal cancer

Plasma acidic glutathione S-transferase (GST-pi) concentrations in 135 patients with gastrointestinal cancer were measured by an improved radioimmunoassay (RIA). Blood was collected into a silicified glass tube containing a specific anticoagulant. The plasma, which was separated rapidly by centrifugation with adequate force, underwent RIA procedures (double antibody and polyethylene glycol separation method). The improved RIA for plasma GST-pi was specific and sensitive. Plasma GST-pi in 75% of patients with esophageal cancer, 64.44% of those with gastric cancer, 74.41% of those with colon cancer, 85.19% of those with hepatocellular carcinoma, and 60.00% of those with pancreatic cancer was elevated higher than 13.99 micrograms/l (mean + 1.96 standard deviation of normal controls). Plasma GST-pi levels in patients with gastric cancer with Stages I-II, III and IV disease increased in proper order. Plasma GST-pi concentrations in 57% of patients with colon cancer decreased to the normal range 14 days after surgery. Plasma GST-pi levels of patients with recurrent colon cancer were higher than of those with primary colon cancer. The procedure for specimen collection must be critical. Plasma GST-pi may be useful in diagnosing gastrointestinal cancer and in monitoring its clinical course.

Separation of multiple forms of acidic glutathione S‐transferase isozymes in a susceptible and a resistant strain of house fly, Musca domestica (L.)

The acidic glutathione S-transferases from a CSMA (susceptible) strain and a Cornell-R (resistant) strain of houseflies were purified and separated utilizing affinity chromatography followed by chromatofocusing. Nine fractions were isolated from each house fly strain. Fraction 1 had the highest 1-chloro-2,4-dinitrobenzene vs. 1,2-dichloro-4-nitrobenzene ratio (CDNB/DCNB ratio) in both strains and the ratio of all the other fractions tended to decrease as the isoelectrical points decreased except for fractions 4 and 9. Most fractions from the CSMA strain had higher CDNB conjugation activities than the fractions from the Cornell-R strain, but all the fractions from the CSMA strain had lower DCNB conjugation activities than fractions from the Cornell-R strain. Steady-state kinetics of all the fractions were examined. The Km values obtained from both strains ranged from 0.36 to 1.12 mM, while the Vmax value ranged from 3.0 to 32.6 mumol/min/mg. In the 100,000 g supernatant, the CDNB specific activities in the CSMA strain was about 1/3 of the activity in the Cornell-R strain but it was about 1.5-fold following affinity chromatography. The specific activity for DCNB measured in the CSMA strain was only 1/5 of the activities of the Cornell-R strain in the 100,000 g supernatant, but was about the same after affinity chromatography. The difference was due to the selectivity of the affinity column used in the current study.

Several Closely Related Glutathione S-Transferase Isozymes Catalyzing Conjugation of 4-Hydroxynonenal Are Differentially Expressed in Human Tissues

A human acidic glutathione S-transferase, hGST 5.8, was isolated from heart, pancreas, and brain by a procedure involving immunoadsorption chromatography on immobilized antibodies raised against mouse mGSTA4-4. The human hGST 5.8 enzymes isolated from these tissues had similar pI (5.8) and subunit Mr (24.5 kDa) values, showed about 17- to 20-fold higher specific activities for 4-hydroxynon-2-enal than that for 1-chloro-2 4-dinitrobenzene, and expressed glutathione peroxidase activity toward phospholipid hydroperoxides. In this respect, the enzymes belong together with rat GST 8-8 and mouse mGSTA4-4 to a subgroup of GSTs involved in the detoxification of lipid peroxidation products. Partial sequencing of CNBr-peptide fragments of hGST 5.8 proteins isolated from various human tissues revealed significant similarity to mGSTA4-4 and the existence of several distinct isoforms differing in their primary structures. These isoforms had similar but nevertheless clearly distinguishable catalytic properties. These results indicate the existence of multiple hGST 5.8-related genes in the humans, which is consistent with our previous studies showing the presence of several closely related genes for the mouse ortholog mGSTA4-4 (Zimniak et al., J. Biol. Chem., 1994, 269, 992-1000).

Site-specific mutagenesis of two histidine residues in fatty acid ethyl ester synthase-III

Human myocardial fatty acid ethyl ester synthase-III is a newly described acidic glutathione S-transferase that metabolizes both ethanol and carcinogens. Structure-function studies have not been performed relating these two distinct enzymatic activities. Since there are only two histidine residues in fatty acid ethyl ester synthase-III (His 72 and His 163), the role of each was examined by site-specific mutagenesis. Fatty acid ethyl ester synthase-III mutagenized at position 72 to contain either Gln, Pro or Ala had less than 5% of control glutathione S-transferase activity but retained fatty acid ethyl ester synthase activity under standard assay conditions. In contrast, substitution of histidine 163 with proline had no effect on glutathione S-transferase activity, but it slightly increased synthase activity. Thus, this study indicates that histidine plays a differential role in fatty acid ethyl ester synthase III depending on the nucleophilic substrate.

1-Chloro-2,4-dinitrobenzene-mediated irreversible inactivation of acidic glutathione S-transferases Inactivation mechanism—A saturation-type or simple second-order kinetic process?

A critical analysis of the inactivation kinetics exhibited by the acidic human glutathione S-transferase (GST) enzymes is presented. Data on the 1-chloro-2,4-dinitrobenzene (CDNB)-facilitated inactivation of human placental GST π have been utilized in conjunction with published inactivation data from the literature to answer the following two questions: (a) do the inactivation kinetics deviate significantly from a simple pseudo first-order model? (b) What is the kinetic mechanism of irreversible electrophilic co-substrate-mediated inactivation of human acidic GSTs? Inactivation of human placental GST π in the presence of 7-aminocephalosporanic acid, a non-electrophilic non-substrate ligand, is characterized and shown to occur via a process analogous to the second mechanism proposed for CDNB inactivation of the enzyme, namely: pH- and [ligand]-independent solvational inactivation.

Purification and Characterization of Acidic Form of Glutathione S-Transferase in Human Fetal Livers: High Similarity to Placental Form1

An acidic form of glutathione S-transferase (GST) was purified from human fetal livers by means of affinity chromatography and chromatofocusing. The major peak of the acidic form of GST was focused between pH 4.8 and 4.9. Judging by SDS-PAGE, the purified acidic GST was apparently homogeneous; the subunit molecular weight was estimated to be 23,000. The acidic GST catalyzed the conjugations of glutathione (GSH) with 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA). The immunochemical properties of the purified acidic GST were indistinguishable from those of human placental GST-pi. The N-terminal amino acid sequence of the acidic GST was identical with that of GST-pi from human placenta. The level of expression of the acidic form of GST was clearly different between human adult and fetal livers as examined on the levels of mRNA and protein.

Purification and characterization of acidic glutathione S-transferase 6 from human brain

An acidic glutathione S-transferase (GST) isoenzyme termed GST6 has been isolated from human brain, characterized and compared with other isoenzymes. The N-terminal amino acid sequence of GST6 was found to be identical with that of GST4 previously purified from human muscle. GST6 cross-reacted with antibody raised against GST4, but not with antisera raised against GST1, GST2 or GST3. The subunit Mr and pI of GST6 were found to be different from those of GST4. The present results indicate that GST6 is another member of the Mu evolutionary class which in man also includes GST1, GST4 and GST5. A minor component that co-purified with GST6 was shown to have an N-terminal sequence similar to, but not identical with, that of GST3. This isoenzyme may be an additional member of the Pi evolutionary class.

Enhanced expression of glutathione S-transferases in colorectal carcinoma compared to non-neoplastic mucosa

Ten paired samples of primary human colorectal carcinoma and adjacent non-neoplastic mucosa were analysed for total glutathione S-transferase (GST) activities as determined by 1-chloro-2,4-dinitrobenzene assays. These tissues were also investigated for the expression of acidic (pi), basic (alpha) and neutral (mu) GSTs using Western blotting procedures and immunohistochemical staining. For each of the paired samples examined the total GST activity was higher in tumour than in adjacent non-neoplastic mucosa. Western blotting, using an antibody against acidic GST also showed strong immunoreactivity in all the samples with more intense reactions in tumour compared to mucosa in nine out of the ten paired samples. Low levels of basic GST were also expressed in all samples of tumour and mucosa. Neutral GST was not detectable in two samples of tumour and corresponding mucosa, but low levels of expression were demonstrated in the remaining eight. Immunohistochemical staining for acidic GST showed a dark brown reaction in all tumour cells; in non-neoplastic mucosa there was positive immunoreactivity for epithelial cells situated deep within the crypts and a negative reaction for surface epithelial cells. Immunohistochemical staining for basic GST was negative except for one sample of tumour and two of mucosa. Neutral GST was expressed only in two samples of tumour and two samples of mucosa. We therefore conclude that there is enhanced expression of GSTs, acidic GST being the predominant form, in tumour compared to normal mucosa, in keeping with a role for GSTs in colonic carcinogenesis and acquired or innate drug resistance.

A comparison of cardiac glutathione S-transferases from wild and domestic animals

1. 1. Cardiac glutathione S-transferases from wild animals; hyena, red fox, porcupine, coypu and mountain gazelle were purified and compared with the enzymes from domestic animals; cow, camel, goat and sheep. 2. 2. By using 1-chloro-2,4-dinitrobenzene as a substrate, domestic hearts expressed higher glutathione conjugating activity than wild animals hearts. 3. 3. In all the studied hearts, the bulk of the activity was associated with near neutral and acidic glutathione S-transferase isozymes with pI values ranging from 4 to 7.4. 4. 4. The enzymes from domestic animals displayed homodimeric structure of 25,000 mol. wt subunit while of the wild animals both hyena and coypu displayed homodimers of 26,500 mol. wt subunit and the rest exhibited heterodimers of 25,000 and 28,000 mol. wt subunits.

Distribution of glutathione S-transferase isoenzymes in human kidney: basis for possible markers of renal injury.

To determine whether the tissue distribution of glutathione S-transferase (GST) isoenzymes could define the precise nature of renal injury, 13 adult kidneys were studied, using specific antibodies raised against purified isoenzymes. Basic GST stained strongly proximal convoluted tubules and some medullary tubules; acidic GST stained strongly distal convoluted tubules and medullary tubules; neutral GST stained similarly to acidic GST, but weaker, and microsomal GST stained glomerular and interstitial endothelium and collecting ducts deep in the medulla, although there was considerable variation in staining intensity among cases. It is suggested that the measurement of these isoenzymes in serum and urine may help to elucidate the localisation of tissue damage, which may be particularly valuable in patients with cyclosporine toxicity following renal transplantation.

Site-directed inactivation of human lung acidic glutathione S-transferase by 1-chloro-2,4-dinitrobenzene in the absence of glutathione

Human lung acidic glutathione S-transferase is irreversibly inhibited by 1-chloro-2,4-dinitrobenzene (CDNB) in the absence of the co-substrate glutathione (GSH). The time-dependent inactivation is pseudo-first-order and demonstrates saturation kinetics, suggesting that inactivation occurs from an EI complex. The K i was 0.14 mM; and k obs was 0.32 min −1 at 0.6 mM CDNB. The enzyme was protected against CDNB inactivation by GSH. The other two classes of glutathione S-transferase, the basic and near-neutral, are not significantly inactivated by CDNB. Incubation with [ 14C]CDNB indicated covalent binding to all three classes of transferase. One peptide fraction was found to be radiolabelled in both the basic and acidic transferases when these were incubated with [ 14C]CDNB and GSH, cleaved with cyanogen bromide, and chromatographed by HPLC. Incubation in the absence of GSH yielded one and two additional labelled peptide fractions for the basic and acidic transferases, respectively. Our results suggest that while CDNB arylates all three classes of human transferases, only the acidic transferase possesses a specific GSH-sensitive CDNB binding site, binding to which leads to time-dependent inactivation.

Radioimmunoassay for Erythrocyte Acidic GSH S-Transf erase

We have developed a RIA for erythrocyte acid glutathione S-transferase (GST), which is immunologically identical to major GSTs from other blood cell components, and measured its serum concentrations in various hematological disorders. In some patients with paroxysmal nocturnal hemoglobinuria, chronic myelomonocytic leukemia, chronic myelocytic leukemia, polycythemia vera and myelofibrosis, the concentrations were high. Very high levels were found in 2 of 3 patients with acute lymphocytic leukemia, while acute myelocytic leukemia exhibited a modest increment. No or little increase was seen in aplastic anemia and myelodysplastic syndrome except chronic myelomonocytic leukemia. It is suggested that the measurement of serum acidic GST may be of use as a clinical marker of increased destruction and/or overproduction of blood cells.

Purification of acidic glutathione S-transferases from human lung, placenta and erythrocyte and the development of a specific radioimmunoassay for their measurement

Human acidic glutathione S-transferases (GST) have been purified from placenta, lung and erythrocytes. The purification protocol resulted in a high yield of pure protein for each tissue, when compared to previous procedures. An apparent subunit M r of 24 800 was calculated for each of the acidic GST and each enzymes had a p(I) of 4.75. No immunochemical differences were detected between the acidic GST isolated from the three tissues. A specific and sensitive radioimmunoassay suitable for the measurement of human acidic GST in plasma or tissues is described.

Electroblotting onto glass-fiber filter from an analytical isoelectrofocusing gel: A preparative method for isolating proteins for N-terminal microsequencing

A new method has been developed for the isolation of proteins for microsequencing. Proteins were separated by isoelectric focusing on polyacrylamide slab gels. Ampholytes in the gel were washed out with 3.5% ( v v ) perchloric acid, and the proteins were electroblotted onto unmodified glass-fiber sheets. The immobilized proteins on the glass-fiber sheet were detected with Coomassie blue dye staining. The protein bands were then excised from the sheet and inserted into a gas phase sequenator for direct sequencing. They could also be extracted with sodium dodecyl sulfate buffer for molecular weight determination. Bovine serum albumin, β-lactoglobulin A, and soybean trypsin inhibitor have been used as standard proteins for the test of this technique. Using this technique, we have determined the partial N-terminal sequence (26 residues) of an acidic (pI 5.6) glutathione S-transferase isolated from the chicken liver.

Studies of the development of basic, neutral and acidic isoenzymes of glutathione S-transferase in human liver, adrenal, kidney and spleen.

The ontogeny of basic, near-neutral and acidic glutathione S-transferase isoenzymes was studied by using chromatofocusing and ion-exchange chromatography. These isoenzyme sets demonstrated tissue-specific patterns of expression. For example, whereas basic isoenzymes were identified in all liver and adrenal cytosols obtained after 10 weeks gestation, these forms were not detected in kidney until 10 weeks post-natal age and in spleen until about 40 weeks post-natal age. Our data indicate that the basic monomers B1 and B2 are present in liver cytosol at 21 weeks gestation. Expression of the near-neutral isoenzymes was usually weak; for example, they were not generally expressed in liver until 30 weeks gestation, and no developmental patterns in their expression could be identified in adrenal, kidney and spleen. The acidic isoenzymes were usually strongly expressed in adrenal, kidney and spleen, although there was a decline in the level of expression in kidney after birth.

The development of glutathione S-transferase and glutathione peroxidase activities in human lung

The development of glutathione S-transferase and glutathione peroxidase activities has been studied in human lung cytosols. Whilst no clear change in glutathione peroxidase activity was identified, expression of the acidic glutathione S-transferase isoenzyme decreased markedly after 15 weeks of gestation so that at birth the level of activity of this isoenzyme was only about 20% of that in samples obtained during the first trimester. Basic glutathione S-transferase isoenzymes were weakly expressed during development and usually comprised less than 10% of cytosolic activity. Ion-exchange studies identified several basic isoenzymes that may correspond to the α, β, γ, δ and ϵ set previously identified in liver. Weak expression of apparently near-neutral isoenzymes was also detected; they were detected in only a few cytosols.

Anion-exchange high-performance liquid chromatography of glutathione s-transferases : Separation of the minor isoenzymes of human erythrocyte, heart and lung

Apparently homogenous preparations of the acidic glutahione S-transferases were subjected to high-performance liquid chromatography over a SynChropak AX-300 anion-exchange column. Results of high-performance liquid chromatography analysis indicated that in each of these tissues the acidic glutathione S-transferases have one major and two minor isoenzymes. All the isoenzymes obtained by high-performance liquid chromatography are dimers of subunits of molecular weight ( M r) 22 500. The major isoenzymes of all the tissues, representing more than 80% of total glutathione S-transferase activity, have similar retention times and comparable kinetic properties. High-performance liquid chromatography profiles of the lung and heart isoenzymes are similar to each other but are significantly different from that of the erythrocytes. These studies provide evidence of microheterogeneity in the acidic isoenzymes of heart, lung and erythrocyte glutathione S-transferasea and provide a rapid and efficient method for separation of minor isoenzymes in more than 95% yield.

Isolation and characterization of the multiple glutathione S-transferases from human liver. Evidence for unique heme-binding sites.

Thirteen forms of glutathione S-transferase were isolated from human liver in high yields by glutathione-affinity chromatography and chromatofocusing. Apparent isoelectric points ranged from 4.9 to 8.9 and included neutral forms. All 13 forms appeared to be identical immunochemically in a quantitative enzyme-linked immunosorbent assay. These forms were immunochemically distinct from the major acidic glutathione S-transferase found in placenta and erythrocyte and were immunochemically distinct from two forms of higher molecular weight glutathione S-transferase found in some but not all liver samples. The 13 forms exhibited similar activities with 1-chloro-2,4-dinitro-benzene as substrate, specific activities of 33-94 mumol/min/mg. Likewise, these forms all exhibited glutathione peroxidase activity with cumene hydroperoxide, specific activities of 1.5-8.3 mumol/min/mg. All 13 forms bound bilirubin with subsequent conformational changes leading to states devoid of transferase activity, a process prevented by the presence of foreign proteins. As hematin-binding proteins, however, these multiple transferases exhibited a very broad range of binding extending from nonbinding to high-affinity binding (KD approximately 10(-8) M). Hematin binding was noncompetitive with transferase activity and did not involve the bilirubin-binding site, suggesting the existence of unique heme-binding sites on these proteins. The two forms of the immunochemically distinct glutathione S-transferases transferases found in some liver samples also exhibited both transferase and peroxidase activities. In addition, they also have separate sites for binding bilirubin and hematin.

Identification of a basic hybrid glutathione S-transferase from human liver. Glutathione S-transferase delta is composed of two distinct subunits (B1 and B2).

The purification of a hybrid glutathione S-transferase (B1 B2) from human liver is described. This enzyme has an isoelectric point of 8.75 and the B1 and B2 subunits are distinguishable immunologically and are ionically distinct. Hybridization experiments demonstrated that B1 B1 and B2 B2 could be resolved by CM-cellulose chromatography and have pI values of 8.9 and 8.4 respectively. Transferase B1 B2, and the two homodimers from which it is formed, are electrophoretically and immunochemically distinct from the neutral enzyme (transferase mu) and two acidic enzymes (transferases rho and lambda). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that B1 and B2 both have an Mr of 26 000, whereas, in contrast, transferase mu comprises subunits of Mr 27 000 and transferases rho and lambda both comprise subunits of Mr 24 500. Antisera raised against B1 or B2 monomers did not cross-react with the neutral or acidic glutathione S-transferases. The identity of transferase B1 B2 with glutathione S-transferase delta prepared by the method of Kamisaka, Habig, Ketley, Arias & Jakoby [(1975) Eur. J. Biochem. 60, 153-161] has been demonstrated, as well as its relationship to other previously described transferases.