Claes Guthenberg

[28] Glutathione transferase (human placenta)

Publisher Summary Glutathione transferases with basic isoelectric points have been purified from the human liver, and an acidic form is purified from human erythrocytes. However, this chapter presents a procedure for the preparation of glutathione transferase from human placenta. Enzyme activity during purification is determined spectrophotometrically at 340 nm by measuring the formation of the conjugate of glutathione and l-chloro-2,4-dinitrobenzene. Purification procedure involve the extraction from the human placenta; the supernatant fraction from Step 1 is chromatographed on a column (12.5 x 80 cm) of Sephadex G-25 and the pooled fractions from Step 2 are applied to a column (9 x 13 cm) of DEAE-cellulose equilibrated with l0 mM Tris-HCl at pH 7.8. The pooled effluent from Step 3 is dialyzed overnight against 3 x 10 liter of 10 mM sodium phosphate buffer; then affinity chromatography is carried on S-hexylglutathione coupled to epoxy-activated sepharose 6B and the pooled fractions from Step 5 are charged onto a column of Sephadex G-75 that is equilibrated and eluted with 10 mM sodium phosphate at pH 6.7, containing 0.1 mM dithioerythritol. The active fractions of effluent are pooled.

Microsomal Glutathione S‐Transferase

Rat liver microsomal glutathione S-transferase was activated with N-ethylmaleimide, solubilized with Triton X-100, and purified by chromatography on hydroxyapatite and CM-Sepharose. A 36-fold purification resulted in a 36% yield, indicating that the glutathione S-transferase accounts for 2.5-3% of the original microsomal protein. The purified protein moved as a band with an apparent molecular weight of 14 000 on sodium dodecyl sulphate gel electrophoresis and appeared to be nearly homogeneous. The complex formed between the purified microsomal glutathione S-transferase and Triton X-100 has a sedimentation coefficient of 3.2 S, a partial specific volume of 0.844 cm3/g, and a Stokes radius of 5.5 nm. The complex has a molecular weight of 127 000 and contains three or four polypeptide chains and 112-134 detergent molecules. Antibodies directed against soluble glutathione S-transferases A, B and C do not react with the purified microsomal enzyme. This finding, together with differences in molecular weight and substrate specificity, demonstrate that the microsomal glutathione S-transferase is an enzyme distinct from the cytosolic glutathione S-transferases.

[62] Glutathione transferases from human liver

Publisher Summary This chapter investigates glutathione transferases derived from human liver. The glutathione transferases are a group of related enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds bearing an electrophilic center. The proteins also act as intracellular binding proteins for a large number of lipophilic substances, including bilirubin. Human glutathione transferases have been purified from liver, erythrocytes, placenta, and lung. A simple and rapid procedure for the purification of basic (α-ɛ) and neutral (μ) glutathione transferases from human liver cytosol is described in the chapter. The enzyme activity during purification is determined spectrophotometrically at 340 nm by measuring the formation of the conjugate of glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The steps of the purification procedure include (1) preparation of cytosol fraction, (2) chromatography on Sephadex G-25, (3) chromatography on DEAE-cellulose, and (4) chromatography on Sephadex G-25.

Rat glutathione transferase 8-8, an enzyme efficiently detoxifying 4-hydroxyalk-2-enals

Rat glutathione transferase 8‐8 is one of the less abundant cytosolic glutathione transferases, accounting for approx. 1% of the total activity with 1‐chloro‐2,4‐dinitrobenzene in liver. The enzyme is eluted at pH 6.3 upon chromatofocusing and has so far been identified in liver, kidney, lung and testis. Characteristic properties include high relative activity with ethacrynic acid (70% of the specific activity with 1‐chloro‐2,4‐dinitrobenzene) and an apparent subunit M r of 24500. The most significant property noted is the high catalytic activity in the conjugation of 4‐hydroxyalk‐2‐enals, major products of lipid peroxidation. The catalytic efficiency with these substrates exceeds corresponding values for all known substrates tested with any glutathione transferase, which suggests that transferase 8‐8 may have evolved to detoxify 4‐hydroxyalk‐2‐enals.

Purification of a new glutathione S-transferase (transferase μ) from human liver having high activity with benzo(α)pyrene-4,5-oxide

A new glutathione S-transferase from human liver has been purified to homogeneity in good yield by use of ion-exchange chromatography on DEAE-cellulose, affinity chromatography on S-hexylglutathione coupled to epoxy-activated Sepharose 6B, and chromatography on hydroxyapatite. This new enzyme, transferase μ, is present in high concentration, but only in some individuals. It has an isoelectric point at about pH 6 to 6.5 and a different substrate specificity than the previously described alkaline transferases α-e from human liver. Especially noteworthy is the finding of high activity against benzo(α)pyrene-4,5-oxide. Glutathione S-transferase μ has about 20-fold higher activity with this substrate than have the alkaline transferases. The most pronounced difference was found with trans-4-phenyl-3-buten-2-one which was >100-fold better as substrate for transferase μ than for the previously described transferases.

Purification of major basic glutathione transferase isoenzymes from rat liver by use of affinity chromatography and fast protein liquid chromatofocusing

Seven major isoenzymes of glutathione transferase with isoelectric points ranging from pH 6.9 to 10 were isolated from rat liver cytosol. The purification procedure included affinity chromatography on immobilized S-hexylglutathione followed by high-performance liquid chromatofocusing. Characteristics, such as physical properties, reactions with antibodies, specific activities with various substrates, kinetic constants, and sensitivities to a set of inhibitors, are given for discrimination and identification of the different isoenzymes. The multiple forms of the enzyme correspond to glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4, and 4-4 in the recently introduced nomenclature [W.B. Jakoby et al. (1984) Biochem. Pharmacol. 33, 2539-2540]. A seventh form appears to be a heterodimeric protein composed of subunit 3 and an as yet unidentified subunit.

GLUTATHIONE TRANSFERASE FROM RAT TESTIS

Publisher Summary Glutathione transferases play an important role in the biotransformation and detoxication of electrophilic xenobiotics. The occurrence of glutathione transferase in animal species is widespread. Several transferase isoenzymes are isolated from rat liver. Six basic transferase isoenzymes in rat hepatic cytosol are characterized as binary combinations of four protein subunits. The presence of glutathione transferase is not restricted to the liver but also is demonstrated in extrahepatic organs. However, in comparison with the liver most other organs show considerably lower activity. One exception is rat testis, which also shows high transferase activity. In contrast with liver, and most other organs, a major part of the glutathione transferase activity in rat testis is borne by an isoenzyme, glutathione transferase 6-6, with an acidic isoelectric point. The purification of this enzyme, which accounts for approximately 50% of the cytosolic glutathione transferase activity, is described in this chapter. In addition to this major acidic isoenzyme, smaller amounts of the basic species, glutathione transferases 2-2, 3-3, 3-4, and 4-4 are identified in testis.

Glutathione S-transferase (transferase π) from human placenta is identical or closely related to glutathione S-transferase (transferase ϱ) from erythrocytes

Abstract Glutathione S-transferase (RX: glutathione R-transferase, EC 2.5.1.18) from human placenta has been purified to homogeneity. This enzyme, transferase π, is an acidic protein (isoelectric point at pH 4.8) composed of two subunits. The molecular weights for the dimer and monomer were determined by independent methods as 47 000 and 23 400, respectively. These properties are not significantly different from those of glutathione S-transferase ϱ from human erythrocytes. Antibodies to transferase π reacted with the enzyme from erythrocytes but not with the basic transferases α-ϵ and the neutral transferase μ isolated from human liver. Antibodies to the latter enzymes did not react with the transferase from placenta. Further similarities between transferases π and ϱ appear in amino acid compositions, kinetic constants and substrate specificities. Both the placental and the erythrocyte enzyme have considerably higher activity with ethacrynic acid than any other of the human glutathione S-transferases. The glutathione S-transferase could be distinguished from two additional acidic glutathione-dependent enzymes, glyoxalase I and selenium-dependent glutathione peroxidase. It is concluded that transferase π from placenta is identical with or very closely related to transferase ϱ from erythrocytes.

Inhibitors for distinction of three types of human glutathione transferase

A set of inhibitors that are useful for distinction of three types of human cytosolic glutathione transferase is presented. The near‐neutral transferase is inhibited most effectively by Cibacron blue (itI50 = 0.05 μM), the acidic transferase by Cibacron blue (itI50 = 0.5 μM), and the basic transferase by tributyltin acetate (itI50 = 0.1 μM). The use of any of these two compounds makes possible differentiation between all three types of human transferase.

Purification of glutathione S-transferases from rat lung by affinity chromatography. Evidence for an enzyme form absent in rat liver

Abstract Glutathione S -transferases from rat lung cytosol were purified about 200-fold in one step by chromatography on S -hexylglutathione bound to epoxy-activated Sepharose 6B. Further purification on hydroxyapatite resolved the lung transferases into five peaks of activity as measured with 1-chloro-2,4-dinitrobenzene as substrate. Three of the peaks were identified with transferases A, B, and C of rat liver on the basis of chromatographic properties, immunochemical reactivity, and substrate specificity. The other two activity peaks were not detectable in liver: one originated from the lung tissue and one appeared to result from blood in the lung.