(6). This steroid derivative also acts as a long-term irreversible anti-glucocorticoid in HTC cells (1-3). The covalently bound steroid-receptor complexes are activated to DNA binding forms (6-8) and then bind to the same long-terminal-repeat regions of mouse mammary tumor virus DNA (9) as the noncovalent complexes.Glutathione S-transferases (glutathione transferase, EC 2.5.1.18) are a family of enzymes that catalyze reactions of glutathione (GSH) with compounds that contain electrophilic centers. Through this type of reaction, the enzyme can detoxify toxic compounds (10-15). Because these enzymes have the capacity to bind a large number of lipophilic substances including endogenous metabolites (e.g., bilirubin, heme, and bile acids), carcinogens, and various other xenobiotics (16-24) an intracellular transport function has also been ascribed to the glutathione-S-transferases. Multiple forms of the rat enzyme arise from dimeric combinations of subunit types designated as Ya, Yb, and Yc (18,(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35).Reports that the Yb form of the protein selectively binds certain steroids (36) prompted this study to explore possible reactions of the protein with steroid affinity probes and to determine whether the protein retains steroid-binding capacity in the presence of other cytosolic proteins, notably glucocorticoid receptors. Evidence is presented that the Yb subgroup of rat liver glutathione-S-transferase is the major cytosolic protein covalently labeled with dexamethasone 21-mesylate.
MATERIALS AND METHODS[6,7-3H(N)]Dexamethasone 21-mesylate in methanol with a specific activity of 48.9 Ci/mmol (1 Ci = 37 GBq) and dexamethasone 21-mesylate powder (unlabeled) were obtained from New England Nuclear. Sephadex G-100, DEAESephacel, protein-A Sepharose CL-6B, and CNBr-activated Sepharose-4B were obtained from Pharmacia. GSH, Smethyl-GSH and dexamethasone were obtained from Sigma and 1-chloro-2,4-dinitrobenzene from Aldrich. Acrylamide and other reagents used for gel electrophoresis were obtained from Bio-Rad.Protein Preparations. Glutathione-S-transferases were prepared as described (35,36). In brief, cytosol prepared from livers of male Sprague-Dawley rats (175-200 g) (105,000 x g supernatants) in 10 mM Tris HCl/1.0 mM EDTA, pH 8.0 (Tris/EDTA buffer) was fractionated by chromatography on Sephadex G-100. The fractions containing glutathione-Stransferase activity were pooled, applied to a lysyl-GSH affinity column (35), and eluted with 10 mM S-methyl glutathione. The multiple forms of the enzyme were resolved by chromatography using 36). Homogeneous preparations of Yb forms with and without bound GSH were separated as described (37).The IgG fraction of rabbit anti-rat Yb-glutathione-S-transferase antisera (36) was purified by a protein A-Sepharose CL-6B column, and it was coupled to CNBr-activated Sepharose 4B to prepare the immunoadsorbant.Enzymatic activities were measured using 1.0 mM 1-chloro-2,4-dinitrobenzene and 1.0 mM GSH as substrates. Absorbance at 343 nm was measured at 25°C in...