Studies on Biochemical Properties of Rat Liver Anionic Glutathione-S-transferase and Its Preferential Binding to Glucocorticoid

A new isozyme of Glutathione-S-transferase (GST) with a more acidic pI (6.7) than other forms of GST hitherto reported was isolated from rat liver cytosol by consecutive chromatographies on a DEAE cellulose column, lysyl-GSH affinity column and Sephadex G-100 column. This anionic form of GST represented approximately one third of the total GST activity in rat liver cytosol. Amino Acid composition, immunological reactivity, enzymatic properties, and secondary structures as measured by circular dichroism of this form were distinct from those of cationic isozymes (GST-AA, GST-B, GST-X). The stoichiometric ratio of high affinity site for bilirubin to the GST molcule differs amongst the cationic isozymes. The anionic form of GST was bound to two bilirubin per one molecule whereas cationic GSTs were bound only to one bilirubin per two subunits. The most distinguished property of anionic GST was its strong affinity for glucocorticoid. The dissociation constant of anionic GST-corticosterone complex is as low as 2.0×10-8M. Corticosterone inhibited the enzyme activity of anionic GST in a noncompetitive fashion with an apparent Ki value of 8.6×10-5M and 1.1×10-6M for 1-chloro-2.4-dinitrobenzene and GSH respectively. The anionic GST-corticosterone complex bound to DNA coupled Sepharose at 25℃ and passed through the column at 4℃. Conversely the complex bound to DEAE cellulose column at 4℃ but passed through at 25℃. These properties of anionic GST are quite similar to those of the glucocorticoid receptor of rat liver cytosol which were reported previously