title: Purification of glutathione S-transferases from rat liver and Walker 256 mammary carcinoma cells by high-performance liquid chromatography and a glutathione affinity column.
abstract: A novel method for the rapid purification of glutathione S-transferases (GST) from tissue and cell culture samples is reported. A high-performance glutathione affinity column was used and produced results comparable to those obtained with classical agarose affinity columns. Experiments with purified rat liver GST standards resulted in 87% recovery of total activity. Sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) of the affinity-purified GST was identical to the GST standard and revealed three major protein bands, corresponding to the Ya, Yb, and Yc subunits. A fourth protein band (relative molecular mass 25,000), migrating slightly faster than the Ya subunit, was present in both the standard and eluted GST samples. This polypeptide was tentatively identified as the Yk subunit. Successful purification from rat liver and Walker 256 rat carcinoma cell cytosols was also performed. Recovery of total GST enzymatic activity from Walker cell and rat liver cytosol was 49 and 58%, respectively. SDS-PAGE of these samples indicated a high degree of purity. This methodology requires less than 1 h and can be performed using small quantities of tissue. These features make this technique applicable to analysis of a broad range of biological applications including human biopsy material for GST content.
authors.affiliation: Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, PA 19111.
authors.forename: F P
authors.initials: FP
authors.lastname: LaCreta
authors.forename: J J
authors.initials: JJ
authors.lastname: Olszewski
authors.forename: K D
authors.initials: KD
authors.lastname: Tew
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D051381:Rats; D014407:Tumor Cells, Cultured / Q000201:enzymology
chemical_list: D005982:Glutathione Transferase