Subunit heterogeneity of cationic human hepatic glutathione S-transferases

We have purified the major reduced glutathione (GSH) S-transferases from 3 apparently normal human livers: Two obtained at surgery and one at autopsy. Purification was by sequential gel filtration, GSH-affinity chromatography, and chromatofocusing. All three livers exhibited the same two major transferase peaks from chromatofocusing at pH 9.0 and 8.7 (designated Cl and C2, respectively) and several (2-4) minor peaks. Another major form (designated Al) from two livers eluted from chromatofocusing at pH 5.4, whereas the major form from the third liver (designated N1) eluted near neutral (pH 6.8). The transferase from erythrocytes eluted at pH 4.6. Isoelectric focusing revealed that the true pi of Al was pH 7.1 indicating that Cl, C2 and Al are all cationic. In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, Cl, C2 and Al exhibited the same single subunit swhereas N1 was different (26, 000). The erythrocyte enzyme had a smaller subunit (23, 500). Urea/SDS-polyacrylamide gel electrophoresis resolved the apparent single subunit of Al, Cl and C2 into two distinct subunits. Cl from all 3 livers was a homodimer of the faster migrating subunit (designated subunit I); C2 was a heterodimer (designated I-II); and Al was a homodimer of the slower migrating subunit (designated subunit II). Hybridization experiments demonstrated that by mixing Cl and Al we could produce C2 whereas dissociation and reassociation of the subunits of C2 generated Cl and Al as well as C2. Rabbit antiserum to Cl recognized Cl and C2, but not Al. Thus, the cationic human hepatic transferases are dimers of two distinct subunits.