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

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Abstract
A previously 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 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 sulfate/polyacrylamide-gel electrophoresis [SDS-PAGE], isoelectric focusing and immunoelectrophoresis. Results from SDS-PAGE and gel filtration indicated that transferase X was a dimer with MW about 45,000 composed of subunits with MW 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 MW, shows that it does not possess subunits Ya or Yc. Glutathione S-transferase X did not catalyze the reaction of menaphthyl sulfate 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. Thus glutathione S-transferase X has properties that differ from those of the known glutathione S-transferases in several respects, although it is closely related to transferases and A and C. However, reduction and structural investigations indicated that glutathione S-transferase X-represents neither an oxidized or a reduced form nor a proteolytic product or precursor of either transferase A or C. This new form appears to be especially important, in that it was substantially more potent in inactivating a diol epoxide, the vicinal dihydrodiol non-bay-region epoxide t-10,11-epoxy-r-8,t-9-dihydroxy-8,9,10,11-tetrahydrobenz[a]anthracene, than any of the other major glutathione S-transferases.

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