Identification of Yb-glutathione-S-transferase as a major rat liver protein labeled with dexamethasone 21-methanesulfonate.

Abstract

(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...

Functional Specific Binding of Testosterone toSchistosoma haematobium28-Kilodalton GlutathioneS-Transferase

“…Mammal GSTs have the ability to bind hormones and particularly sexual steroids, to influence their transport, metabolism, and action (13,16). Direct binding of testosterone for mammal GST with moderate affinity (10 (16).…”

“…Mammal GSTs are also involved in the intracellular transport of a variety of endogenous metabolites, drugs, and hormones by their abilities to bind these substances (16). Particularly, GSTs are glucocorticoid-binding proteins and, thereby, may influence transport, metabolism, and action of steroids (13). It has also been demonstrated that testosterone and progesterone have the ability to bind mammal GSTs with moderate (10…”

Glutathione-S-transferase-P1 I105V polymorphism and response to antenatal betamethasone in the prevention of respiratory distress syndrome

“…The GST family of enzymes, and in particular the GST-P1 isoform, is localized mainly in the cytotrophoblast at all stages of pregnancy, and its expression increases WT Wild type with development [51]. These enzymes bind various ligands such as steroids [20][21][22] and may play a role in the clinical response to GCs in acute lymphoblastic leukemia [25,52]. Phenotypically relevant genetic polymorphisms have been identified also in some human cytosolic GST isoenzymes, and in particular, in families M1, P1, and T1.…”

“…These enzymes have previously been shown to bind to GCs and to play a role in the metabolism and response to these hormones [20][21][22][23]. Phenotypically relevant genetic polymorphisms for some GST subclasses (GST-M1, GST-T1, and GST-P1) have been related to the outcome of several diseases, including acute lymphoblastic leukemia and solid tumors [24], and to response to GCs [25].…”

Differential regulationof glutathione S-transferase Yb1 mRNA levels in rat prostate, liver and brain by androgen

“…In addition, the transferases bind a number of hydrophobic compounds such as heme, bilirubin and dexamethasone. Their expression can be induced by various xenobiotics such as transstilbene oxide [9,10] and for many toxic xenobioties, glutathione S-conjugate formation represents a detoxication pathway [7,8]. The GSTs are either homodimers or heterodimers composed of at least eight subunits (Ya, Yb1, Yb2, Yb3, Yc, Yk, Yn and Yp) which are expressed tissue-specifically and can be distinguished according to their substrate specificity.…”