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 C1 and C2, respectively) and several (2-4) minor peaks. Another major form (designated A1) 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 A1 was pH 7.1 indicating that C1, C2 and A1 are all cationic. In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, C1, C2 and A1 exhibited the same single subunit (25,000) whereas 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 A1, C1 and C2 into two distinct subunits. C1 from all 3 livers was a homodimer of the faster migrating subunit (designated subunit I); C2 was a heterodimer (designated I-II); and A1 was a homodimer of the slower migrating subunit (designated subunit II). Hybridization experiments demonstrated that by mixing C1 and A1 we could produce C2 whereas dissociation and reassociation of the subunits of C2 generated C1 and A1 as well as C2. Rabbit antiserum to C1 recognized C1 and C2, but not A1. Thus, the cationic human hepatic transferases are dimers of two distinct subunits.
D000596:Amino Acids / Q000032:analysis; D002021:Buffers; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D002021:Buffers; D005982:Glutathione Transferase