Glutathione S-transferase AA Research Articles

Purification of bile acid-binding proteins from rat hepatic cytosol. Use of a photoaffinity label to detect novel Y′ binders

A 125I-labelled photolabile derivative of cholic acid has been used to investigate the organic anion-binding Y′ fraction from rat liver, prepared by the method of Sugiyama, Y., Yamada, T. and Kaplowitz, N. (1982) Biochimica Biophysica Acta, 709, 342–352. The use of this photoaffinity probe led to the discovery of previously undescribed bile acid-binding proteins. A comprehensive purification scheme for the Y′ proteins which allows the isolation of these novel binding species is described. Electrophoretic analysis shows that the Y′ binders can be divided into two groups. The proteins in group 1 are dimeric and the 5B, 6E and 7F binding species consist of subunits with approximate molecular masses of 19.6, 15.6 and 14.9 kDa, respectively. The group 2 binding proteins, 5C, 5D and 8C, are monomeric and have molecular masses of approximately 36.2, 36.2 and 33 kDa, respectively. Calculation of the incorporation of 125I by these proteins showed that the group 1 proteins displayed a significantly greater specific incorporation of radioactivity than group 2. The specificity of 125I-labelled 3β-azidocholylhistamine is further demonstrated by analysis of tryptic digests of photoaffinity labelled Y′ binders and glutathione S-transferases AA, A, D and F by reverse-phase high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). The majority of the radioactivity was shown to be incorporated into a single component, which was not coincident with the free photoaffinity label.

The glutathione S-transferases in selenium and vitamin E deficiency.

Preliminary experiments confirmed the work of others showing that the total glutathione peroxidase (GSH-px) activity of rat liver supernatant fraction may be resolved into two peaks of activity (peaks I and II) by gel filtration, and that peak I is the selenium-containing enzyme and peak II is another peroxidase indistinguishable from glutathione S-transferase (GST). In selenium and vitamin E deficiency, the total activity of the GSH-px became very low, and the total activity of GST with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate was enhanced. Study of the time course of these changes as deficiency progressed indicated that the stimulus for the rise in GST (CDNB) activity was the fall in GSH-px activity which preceded it. The peroxidase activity of GST was found to reside only in the GST AA, B and B2 forms of the enzyme, which were shown to be respectively a homodimer of the Yc subunit, a homodimer of the Ya subunit and a heterodimer of the YaYc subunit. As vitamin E and selenium deficiency progressed, the B2 and AA forms of the enzyme showed enhanced activity, which was interpreted as implying that the Yc subunit of the enzyme becomes enriched as a consequence of the withdrawal of selenium from the animal's diet. Densitometric measurements of the Yc and Ya subunits confirmed that the amount of the Yc subunit was nearly doubled in selenium deficiency, relative to the Ya subunit.

Chloroform-induced alteration of glutathione s-transferase activity

The effect of chloroform treatment on the hepatic glutathione S-transferases was studied in phenobarbital-treated rats. The apparent isozymic composition of glutathione S-transferases in hepatic cytosol was changed after chloroform treatment. Glutathione S-transferases AA, A, B, C, and D + E were observed in hepatic cytosol from untreated rats; in contrast, the catalytic activity associated with basic glutathione S-transferases, such as AA, A, B, and C, decreased with time after chloroform treatment. Glutathione S-transferase B was not detectable 2 hr after chloroform treatment, and glutathione S-transferases AA and C were scarcely detectable after 5 hr. Twenty-four hours after chloroform treatment, glutathione S-transferases A and C were clearly detectable as was D + E and a small amount of B. Hepatic cytosolic glutathione S-transferase activity was decreased by chloroform treatment, and reached a minimum at 5 hr after treatment. Corresponding to the decrease of hepatic cytosol glutathione S-transferase activity, serum glutathione S-transferase activity was elevated maximally 5 hr after chloroform treatment and returned to almost normal by 24 hr. Treatment of rats with SKF 525-A or cysteine inhibited the chloroform-induced elevation of serum glutathione S-transferase activity. The chromatographic properties of the gluathione S-transferases present in serum were similar to glutathione S-transferase D + E. Furthermore, after incubation of partially purified cytosolic glutathione S-transferases with chloroform in the presence of hepatic microsomes and NADPH, only transferase D + E was detected. The addition of bilirubin to partially purified cytosolic glutathione S-transferase decreased the basic character of glutathione S-transferases B and C. In conclusion, chloroform caused a release of hepatic cytosolic glutathione S-transferases into serum. Both the active metabolite of chloroform, which was produced by the microsomal cytochrome P-450 system, and bilirubin, which was increased by chloroform treatment, played roles in altering the properties of the glutathione S-transferases.

Purification and characterization of a new cytosolic glutathione S-transferase (glutathione S-transferase X) from rat liver

A hitherto unknown cytosolic glutathione S-transferase from rat liver was discovered and a method developed for its purification to apparent homogeneity. This enzyme had several properties that distinguished it from other glutathione S-transferases, and it was named glutathione S-transferase X. The purification procedure involved DEAE-cellulose chromatography, (NH4)2SO4 precipitation, affinity chromatography on Sepharose 4B to which glutathione was coupled and CM-cellulose chromatography, and allowed the isolation of glutathione S-transferases X, A, B and C in relatively large quantities suitable for the investigation of the toxicological role of these enzymes. Like glutathione S-transferase M, but unlike glutathione S-transferases AA, A, B, C, D and E, glutathione S-transferase X was retained on DEAE-cellulose. The end product, which was purified from rat liver 20 000 g supernatant about 50-fold, as determined with 1-chloro-2,4-dinitrobenzene as substrate and about 90-fold with the 1,2-dichloro-4-nitrobenzene as substrate, was judged to be homogeneous by several criteria, including sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, isoelectric focusing and immunoelectrophoresis. Results from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration indicated that transferase X was a dimer with Mr about 45 000 composed of subunits with Mr 23 500. The isoelectric point of glutathione S-transferase X was 6.9, which is different from those of most of the other glutathione S-transferases (AA, A, B and C). The amino acid composition of transferase X was similar to that of transferase C. Immunoelectrophoresis of glutathione S-transferases A, C and X and precipitation of various combinations of these antigens by antisera raised against glutathione S-transferase X or C revealed that the glutathione S-transferases A, C and X have different electrophoretic mobilities, and indicated that transferase X is immunologically similar to transferase C, less similar to transferase A and not cross-reactive to transferases B and E. In contrast with transferases B and AA, glutathione S-transferase X did not bind cholic acid, which, together with the determination of the Mr, shows that it does not possess subunits Ya or Yc. Glutathione S-transferase X did not catalyse the reaction of menaphthyl sulphate with glutathione, and was in this respect dissimilar to glutathione S-transferase M; however, it conjugated 1,2-dichloro-4-nitrobenzene very rapidly, in contrast with transferases AA, B, D and E, which were nearly inactive towards that substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of chlorophenoxyalkyl acid herbicides with rat-liver glutathione S-transferases

The in vitro interaction of four chlorophenoxyalkyl (CPA) acid herbicides with rat-liver glutathione S-transferase (GST) was studied using reduced glutathione and 1-chloro-2,4-dinitrobenzene as substrates. Inhibition of GST activity by the CPA acids in crude extracts was dose dependent. Ring substitution and side-chain length were shown to be of importance in determining the extent of GST inhibition. While GST AA, an isoenzyme of GST, was stimulated by two CPA acids, each of the other GST isoenzymes (A, B, C, E and M) was inhibited, to different degrees. Kinetic studies revealed a mixed type inhibition of the isoenzymes. Conjugates of CPA acids with glutathione were not formed. These results indicate that CPA acids interact with GST by binding directly to these proteins, possibly at a different locus from that of the substrate. The binding of CPA acids to GST may, therefore, have a protective function against these herbicides.

Soluble glutathione s-transferase isoenzymes in rat brain

The soluble glutathione S-transferase (GST) isoenzymes in rat brain were investigated using 1-chloro-2,4-dinitrobenzene (CDNB) as the second substrate. The percentages of the different CDNB-GST isoenzymes found were: anionic GST: 3.7%, GST D + E: 35.3%, GST C: 27.9%, GST B: 0.5%, GST A: 13.9% and GST AA: 18.6%. The percentages of isoenzymes are quite different from those measured in liver, testis and prostate. An important detoxication role of GST in brain is suggested.

Isolation of the major cyanogen bromide fragment of the a- and c-subunits of glutathione S-transferases AA and B and ligandin

Isolation of the major cyanogen bromide fragment of the a- and c-subunits of glutathione <i>S</i>-transferases AA and B and ligandin

Prostatic glutathione S-transferase in rat, guinea pig and rabbit

The occurrence of soluble glutathione S-transferase (GST) in the prostate of rat, guinea pig and rabbit is reported. With 1-chloro-2,4-dinitrobenzene (CDNB) as the second substrate a specific activity of 59 nmol product/min/mg protein was found in the rat. A value of approx. 2 x and 25 x higher was found for guinea-pig and rabbit respectively. GST activity was also demonstrated with 3 other substrates. GST C was the most abundant CDNB—GST isoenzyme (76%) in rat prostate, followed by GST A (17%) and GST AA (7%), significant amounts of other isoenzymes not being found. The presence of GST in the prostate suggests that these enzymes may play a role in the metabolism and detoxification of electrophilic xenobiotics.

A study of the structures of the YaYa and YaYc glutathione S-transferases from rat liver cytosol Evidence that the Ya monomer is responsible for lithocholate-binding activity

The two dimeric lithocholic acid-binding proteins previously identified as ligandin (YaYa) and glutathione S-transferase B (YaYc) were isolated from rat liver cytosol. These proteins have molecular weights of 44000 and 47000 respectively. The recovery of these two proteins from liver was not affected by the addition of the proteinase inhibitor Trasylol. No spontaneous interconversion between these two proteins was observed on storage. YaYa and YaYc proteins yielded peptides of identical molecular weight after limited digestion with Staphylococcus aureus V8 proteinase. Analytical and preparative tryptic-digest peptide 'maps' showed that all the soluble peptides obtained from YaYa protein were also recovered from YaYc protein. Approximately six extra soluble peptides, which were not recovered from YaYa protein, were obtained from the tryptic digest of YaYc protein. Subdigests of the insoluble tryptic-digest 'cores' also resulted in the recovery of identical peptides from both proteins. Evidence is presented that the Ya subunit possessed by both proteins is identical; glutathione S transferase B is a hybrid of ligandin and glutathione S-transferase AA. The Ya monomer is responsible for lithocholate binding.

The identity of the c-subunits of glutathione S-transferases AA and B

The identity of the c-subunits of glutathione <i>S</i>-transferases AA and B

Multiplicity of sulfobromophthalein-binding proteins in Y-fraction from rat liver.

Several species of the sulfobromophthalein-binding proteins were isolated from the Y-fraction of rat liver cytosol. Each of these proteins, designated as Cv, C1, C2 and C3 according to the elution order from CM-Sephadex column, migrated to the same position as a single protein band in dodecylsulphate electrophoresis. They are basic proteins of approximately 46000 daltons, composed of two subunits of apparently equal size, and similar to the glutathione S-transferase AA, A, B (ligandin), C purified by Jakoby et al. Each of the Cv, C1 and C2 protein forms 1 : 1 primary complex with BSP, with the binding constant of about 5×106 M-1 or more. The C1 protein has a somewhat higher binding constant than the others. The binding constant was little influenced by the purification. The binding constants of these proteins for other organic anions were determined by the competitive technique using 1-anilino-8-naphthalenesulfonate. These proteins show broad binding specificity for various organic anions which are all known to be rapidly transported into the liver. From these results, it is concluded that the binding of BSP by the Y-fraction is not due to a single protein like ligandin.

Glutathione S-transferase AA from rat liver

Glutathione S-transferase AA from rat liver was purified to apparent homogeneity as judged by gel filtration and gel electrofocusing. The protein has an isoelectric point near pH 9.9 and a molecular weight of 45,000 and is composed of two apparently identical subunits. The enzyme is most active with 1-chloro-2,4-dinitrobenzene and glutathione as substrates. The catalytic properties of transferase AA are very similar to those of transferase B although the two proteins differ in their ability to bind bilirubin and other ligands, in their amino acid composition, and in their immunological properties. When mixtures of transferase AA with other purified glutathione S-transferases were denatured in 6 m guanidine hydrochloride and then renatured by dilution, no hybrids were formed as judged by gel electrofocusing. Isolation of the enzymes from a single rat liver revealed the presence of each of the known glutathione transferases.

Binding of nonsubstrate ligands to the glutathione S-transferases.

Fluorescence spectroscopy and inhibition kinetics were used to quantitate the affinity of nonsubstrate ligands for the rat liver glutathione S-transferases AA, A, B, and C in the presence of glutahione. The dissociation constants KD, for ligands such as bilirubin, indocyanine green, and hematin were determined by measuring the decrease in the intrinsic fluorescence of the proteins attendant on the addition of ligand. A second technique, used for compounds which absorb strongly at the excitation maxima of tryptophan, was to utilize 8-anilinonaphthalen sulfonate in the formation of protein complex fluorescing at a higher wavelength. The quenching of this complex allowed the determination of the dissociation constants for ligands such as 3,6-dibromosulfophthalein and cephalothin. These data indicate that all four proteins bind these ligands but do so with different affinities. The bilirubin-induced decrease in fluorescence was used to estimate the stoichiometry of binding as 1.2 mol of bilirubin bound/mol of transferase B and 0.7 mol/mol of transferase C. All of the ligands examine are inhibitors of catalytic activity, as tested in a standard assay with GSH and 1-chloro-2,4-dinitrobenzene as substrates. From these studies we conclude that these proteins have a broad specificity not only for their substrates, but for the binding of nonsubstrate ligands as well.