Identification of a glutathione S-transferase without affinity for glutathione sepharose in human kidney

Summary. To identify kidney glutathione S-transferase (GST) isoenzyme, which does not bind to glutathione affinity column, biochemical characterization was performed by using an array of substrates and by measuring sensitivity to inhibitors. Immunological characterization was done by immunoblotting. Affinity flow-through GST exhibited activity towards 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and cumene hydroperoxide, typical class α substrates and high sensitivity towards hematin, an α class inhibitor. It cross-reacted with antibodies against α class GST. Affinity flow-through GST in human kidney is an α class member.     

Evidence for a common high affinity binding site on glutathione S-transferase B for lithocholic acid and bilirubin

Binding of lithocholic acid, bilirubin, and gossypol to glutathione S-transferase B (ligandin or transferase YaYc) was compared using four methods. Tryptophan quenching revealed a single high affinity site for bilirubin and gossypol but could not be used for lithocholic acid. Both displacement of the fluorescent probe, 1-anilino-8-naphthalenesulfonate, and spectral changes induced by bilirubin binding demonstrated a common high affinity site for which all three ligands compete. Similar results were obtained by equilibrium dialysis. The dissociation constants for the binding of both bilirubin and lithocholic acid were comparable with the various methods (range 0.2-0.7 microM). Thus, lithocholic acid and bilirubin share a high affinity binding site on gluthathione S-transferase B that appears to be separate from the binding site for substrates.     

Protection of glutathione S-transferase from bilirubin inhibition

Inhibition of the enzyme activity of glutathione S-transferase (GST) by a physiological concentration of bilirubin was studied using various substrates. When rat liver cytosol was used as an unfractionated GST, its GSH-conjugation activity toward 1-chloro-2,4-dinitrobenzene was decreased to one-half by bilirubin, while the activity toward 1,2-dichloro-4-nitrobenzene, p-nitrobenzyl chloride, or 1,2-epoxy-(p-nitrophenoxy)propane and also the non-selenium dependent GSH-peroxidase activity toward cumene hydroperoxide (CHPx activity) were hardly affected under the same conditions. In contrast, bilirubin inhibited each of the purified GST isozymes and no remarkable difference in bilirubin inhibition was observed with any of the substrates tested. From the chromatographic analysis of the cytosol incubated with [3H]bilirubin, it was found that a major part of the added bilirubin binds to subunit 1 (Ya) of GST isozyme, leaving not only the conjugation activity derived from 3-4 type GST but also the CHPx activity of subunit 2 (Yc) quantitatively intact. The bilirubin inhibition of both the conjugation activity of GST 3-4 and the CHPx activity of GST 2-2 was prevented almost completely by addition of a 3-fold molar excess of GST 1-1. From these results, it was assumed that the enzyme activities of both 3-4 type GSTs and subunit 2 (Yc) were protected from the inhibitory action of bilirubin by the scavenger effect of subunit 1 (Ya).     

Multiple forms of glutathione S-transferase from pig liver--reaction with methyl linoleate hydroperoxides

1. Seven isoenzyme forms of glutathione S-transferase were purified from pig liver. 2. The most basic isoenzyme reduced methyl 13-hydroperoxy-cis-9,trans-11-octadecadienoate in the absence of detergent at a higher rate (0.3 mumol/min/mg protein) than predicted from substrate solubility. 3. This demonstrates that glutathione transferase possesses some surface acting character for neutral lipid hydroperoxides.     

Acidic and basic forms of glutathione S-transferases from human placenta and comparison with human kidney glutathione S-transferase

Glutathione S-transferase (GSH-transferase) was purified from human placenta and kidney by affinity chromatography on S-glutathione-carbamidomethyl-epsilon-aminolysyl-Sepharose CL 4B and gel filtration chromatography on Sephades G-75. Electrophoretically pure enzyme with the specific activities of 50.7 and 55.9 U/mg, respectively, were obtained. In addition to the known acidic isoenzyme from human placenta (isoelectric point, pI, 4.5), we describe here for the first time the presence of 6 basic forms with pI values between 8.0 and 9.0. The kidney GSH-transferase contained 2 acidic forms with isoelectric points at 4.6 and 4.65, and 6 basic forms with pI values between 8.7 and 9.4. The basic and acidic isoenzymes from placenta were separated by ion exchange chromatography on Sephadex DEAE A-25. The acidic form accounted for 36% of the total GSH-transferase activity from placenta. Antibodies against the kidney enzyme were raised in rabbit. Total cross-reactivity of placental GSH-transferase with antikidney-GSH-transferase antibodies was obtained, suggesting that the kidney and placental enzymes are immunologically closely related.     

Erythrocyte glutathione S-transferase. Electrophoretic identification of two enzyme forms.

Starch-gel electrophoresis was used to demonstrate two forms of glutathione S-transferase in human erythrocytes. Whereas considerable inter-individual differences in enzyme activity and electrophoretic patterns were detected, intra-individual differences were small.     

Variability of glutathione S-transferase of human erythrocytes.

Glutathione transferase (GST) of human erythrocytes, while homogeneous upon electrophoresis, varied more than sixfold in amount in individuals. The levels of this enzyme were found to be inherited, but no practical way was devised to use this enzyme as a genetic marker.     

Purification and physical characterization of glutathione S-transferase K. Differential use of S-hexylglutathione and glutathione affinity matrices to isolate a novel glutathione S-transferase from rat liver.

A novel hepatic enzyme, glutathione S-transferase K, is described that, unlike previously characterized transferases, possesses little affinity for S-hexylglutathione-Sepharose 6B but can be isolated because it binds to a glutathione affinity matrix. A purification scheme for this new enzyme was devised, with the use of DEAE-cellulose, S-hexylglutathione-Sepharose 6B, glutathione-Sepharose 6B and hydroxyapatite chromatography. The final hydroxyapatite step results in the elution of three chromatographically interconvertible forms, K1, K2 and K3. The purified protein has an isoelectric point of 6.1 and comprises subunits that are designated Yk (Mr 25,000); during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it migrates marginally faster than the Ya subunit but slower than the pulmonary Yf monomer (Mr 24,500). Transferase K displays catalytic, immunochemical and physical properties that are distinct from those of other liver transferases. Tryptic peptide maps suggest that transferase K is a homodimer, or comprises closely homologous subunits. The tryptic fingerprints also demonstrate that, although transferase K is structurally separate from previously described hepatic forms, a limited sequence homology exists between the Yk, Ya and Yc polypeptides. These structural data are in accord with the immunochemical results presented in the accompanying paper [Hayes & Mantle (1986) Biochem. J. 233, 779-788].     

Secretion and affinity purification of glutathione S-transferase fusion proteins from yeast

Summary Sequences encoding GST-fusion proteins were cloned into the Saccharomyces cerevisiae secretion vector, pYEX-S1, to direct secretion into the culture medium. GST and metallothionein fused to GST were secreted successfully and the fusion proteins purified. With several other GST-fusion proteins however, the proteins were retained inside the cell, indicating limitations to the types of proteins that can be secreted from yeast.     

Distribution of glutathione S-transferase isoenzymes in human ovary.

Glutathione S-transferases (GST) are drug-metabolizing and detoxification enzymes involved in the intracellular transport and metabolism of steroid hormones. We studied expression of pi, alpha, mu and microsomal GST by immunohistochemistry in normal human ovaries at different stages of the menstrual cycle and pregnancy and after the menopause. Antibodies were raised in rabbits to purified GST subunits and formalin-fixed, paraffin-embedded sections were studied using the peroxidase-antiperoxidase method. Staining density was graded from very strong to negative. All four isoenzymes were identified in the ovary and their distribution was heterogeneous. The staining pattern of follicles varied with the stage of the menstrual cycle for each isoenzyme. All the ovaries contained abundant GST pi in stroma. GST alpha is closely associated with the glutathione-dependent enzyme delta-5,3-ketosteroid isomerase, which catalyses the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione. GST alpha was localized to the steroid-producing cells and thus may be useful in studying ovaries in conditions where there are assumed alterations in steroid production.     

alpha-Tocopherol inhibits human glutathione S-transferase pi

alpha-Tocopherol is the most important fat-soluble, chain-breaking antioxidant. It is known that interplay between different protective mechanisms occurs. GSTs can catalyze glutathione conjugation with various electrophiles, many of which are toxic. We studied the influence of alpha-tocopherol on the activity of the cytosolic pi isoform of GST. alpha-Tocopherol inhibits glutathione S-transferase pi in a concentration-dependent manner, with an IC(50)-value of 0.5 microM. At alpha-tocopherol additions above 3 microM there was no GST pi activity left. alpha-Tocopherol lowered the V(max) values, but did not affect the K(m) for either CDNB or GSH. This indicates that the GST pi enzyme is noncompetitively inhibited by alpha-tocopherol. An inhibition of GST pi by alpha-tocopherol may have far-reaching implications for the application of vitamin E.     

Immunohistochemistry of omega class glutathione S-transferase in human tissues.

Omega class glutathione transferase (GSTO) has been recently described in a number of mammalian species. We used immunohistochemistry to determine the cellular and tissue distribution of GSTO1-1 in humans. Expression of GSTO1-1 was abundant in a wide range of normal tissues, particularly liver, macrophages, glial cells, and endocrine cells. We also found nuclear staining in several types of cells, including glial cells, myoepithelial cells of the breast, neuroendocrine cells of colon, fetal myocytes, hepatocytes, biliary epithelium, ductal epithelium of the pancreas, Hoffbauer cells of the placenta, and follicular and C-cells of the thyroid. These observations and the known activity of GSTO1-1 suggest biological functions that are not shared with other GSTs.     

Crystallization of glutathione S-transferase from human placenta

Crystals of an acidic pi class glutathione S-transferase from human placenta have been obtained by the hanging drop method using ammonium sulphate as a precipitant. The crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2) with cell dimensions a = b = 60.1 A, c = 244.0 A. They contain a dimer in the asymmetric unit and diffract to a resolution of 2.7 A.     

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 alpha, beta, gamma, delta and epsilon set previously identified in liver. Weak expression of apparently near-neutral isoenzymes was also detected; they were detected in only a few cytosols.     

Tocopherol esters inhibit human glutathione S-transferase omega

Human glutathione S-transferase omega 1-1 (hGSTO1-1) is a newly identified member of the glutathione S-transferase (GST) family of genes, which also contains alpha, mu, pi, sigma, theta, and zeta members. hGSTO1-1 catalyzes the reduction of arsenate, monomethylarsenate (MMA(V)), and dimethylarsenate (DMA(V)) and exhibits thioltransferase and dehydroascorbate reductase activities. Recent evidence has show that cytokine release inhibitory drugs, which specifically inhibit interleukin-1b (IL-1b), directly target hGSTO1-1. We found that (+)-alpha-tocopherol phosphate and (+)-alpha-tocopherol succinate inhibit hGSTO1-1 in a concentration-dependent manner with IC50 values of 2 microM and 4 microM, respectively. A Lineweaver-Burk plot demonstrated the uncompetitive nature of this inhibition. The molecular mechanism behind the inhibition of hGSTO1-1 by alpha-tocopherol esters (vitamin E) is important for understanding neurodegenerative diseases, which are also influenced by vitamin E.     

Preferential binding of steroids by anionic forms of rat glutathione S-transferase

Rat liver glutathione S-transferases with isoelectric points near 6.7 were resolved from more basic forms of the protein. This anionic fraction represented about 30% of the total activity in liver with 1-chloro-2,4-dinitrobenzene and was the preponderant form utilizing trans-4-phenyl-3-butene-2-one as a substrate. The anionic transferases are dimeric proteins composed of two subunits designated as Yb and were distinguished from the cationic transferases on the basis of structural, immunological, and binding properties. Amino acid compositions and immunological properties of the anionic protein were similar to those of glutathione S-transferases A and C. The anionic forms had substantially less ordered secondary structure than cationic forms composed of subunits Ya and Yc. Stoichiometric ratios of two high affinity binding sites per dimer, also differentiated between the anionic and all of the cationic transferases which bind only a single mole of ligand. Affinity matrices composed of corticosterone or cholate, and circular dichroism methods, were used to demonstrate selective binding of steroids and bile acids to the anionic glutathione S-transferases. Glucocorticoids and progestins were shown to bind with high affinity whereas estrogens were bound at distinct lower affinity sites. In contrast to the cationic transferases, glutathione had no effect on binding of the steroids to the anionic forms, which suggested that these proteins have the capacity to bind these substances even in a milieu with high concentrations of glutathione.     

Distinctions between the multiple cationic forms of rat liver glutathione S-transferase

Three cationic glutathione S-transferase forms isolated from rat liver were characterized as dimers that originated from different combinations of two subunit types, Ya and Yc. The cationic forms were purified using lysyl glutathione affinity matrices and were chromatographically resolved from anionic glutathione S-transferases that contain Yb subunits. The three classes of cationic transferase exhibited similar specific activities with 1-chloro-2,4-dinitrobenzene as a substrate, all forms cross-reacted with antibodies to glutathione S-transferase B, and all had comparable secondary structures and tryptophan fluorescence properties. In spite of those similarities, the Yc-containing forms were clearly distinguishable from Ya forms on the basis of characteristic differences in circular dichroic patterns associated with their aromatic side chains. All cationic transferases bound bilirubin with stoichiometric ratios of 1 mol/dimeric protein molecule, but discrete differences in mode of binding were ascribed to forms containing Ya subunits as compared to Yc dimers. Binding to Yc forms was of lower affinity and may be associated with the catalytic region of the protein since glutathione effectively displaced bilirubin from the Yc component.     

Multiple Ya subunits of glutathione S-transferase detected by monoclonal antibodies

Monoclonal antibodies to ligandin (YaYa) and glutathione (GSH) S-transferase B (YaYc) were produced by hybridomas derived from the fusion of mouse myeloma cells and spleen cells of mice immunized with the YaYa or YaYc proteins, respectively. Enzyme-linked immunosorbent assay was used to screen for antibody-producing clones. Immunoblotting of the subunits of transferase B, ligandin, and another GSH S-transferase containing Yb subunits showed that the monoclonal antibodies produced by two anti-YaYa subclones recognized the Ya subunits of both ligandin and transferase B, but they did not bind Yc or Yb subunits. It was also revealed that antibodies produced by several anti-YaYc subclones recognized the Yc subunit, but not the Ya subunit of the antigen which was used for the immunization of the mice. However, these monoclonal antibodies did bind the Ya subunit of ligandin. These results indicate that the Ya subunits of GSH S-transferase B and of ligandin do share at least one common determinant. However, these two Ya subunits are structurally distinct as evidenced by their differences in binding by monoclonal anti-YaYc antibodies.     

3-Methyleneoxindole: an affinity label of glutathione S-transferase pi which targets tryptophan 38.

The compound 3-methyleneoxindole (MOI), a photooxidation product of the plant auxin indole-3-acetic acid, functions as an affinity label of the dimeric pi class glutathione S-transferase (GST) isolated from pig lung. MOI inactivates the enzyme to a limit of 14% activity. The k for inactivation by MOI is decreased 20-fold by S-hexylglutathione but only 2-fold by S-methylglutathione, suggesting that MOI does not react entirely within the glutathione site. The striking protection against inactivation provided by S-(hydroxyethyl)ethacrynic acid indicates that MOI reacts in the active site region involving both the glutathione and the xenobiotic substrate sites. Incorporation of [(3)H]MOI up to approximately 1 mol/mol of enzyme dimer concomitant with maximum inactivation suggests that there are interactions between subunits. Fractionation of the proteolytic digest of [(3)H]MOI-modified GST pi yielded Trp38 as the only labeled amino acid. The crystal structure of the human GST pi-ethacrynic acid complex (2GSS) shows that the indole of Trp38 is less than 4 A from ethacrynic acid. Similarly, MOI may bind in this substrate site. In contrast to its effect on the pi class GST, MOI inactivates much less rapidly and extensively alpha and mu class GSTs isolated from the rat. These results show that MOI reacts preferentially with GST pi. Such a compound may be useful in novel combination chemotherapy to enhance the efficacy of alkylating cancer drugs while minimizing toxic side effects.