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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.

ABSTRACT: 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].

SUBMITTER: Hayes JD

PROVIDER: S-EPMC1153100 | biostudies-other | 1986 Feb

REPOSITORIES: biostudies-other

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Project description: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)

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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.

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