The Identity of Glutathione S -Transferase B with Ligandin, a Major Binding Protein of Liver

Abstract

Evidence is presented that ligandin, an intracellular protein involved in the binding of such anions as bilirubin, indocyanine green, and penicillin, is identical to glutathione S-transferase B (EC 2.5.1.18), an enzyme catalyzing the conjugation of glutathione with such electrophiles as 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, iodomethane, ethacrynic acid, and bromosulfophthalein. The proteins, isolated by distinct methods, have the same specificity for substrates and for ligands, react in identical fashion to antibody pioduced against ligandin, bear entirely sinlilar physical characteristics and amino acid composition, and are both induced in response to phenobarbital. Indocyanine green, one of the ligands that is not effective as a substrate, was showh to competitively inhibit the conjugation reaction. It is suggested that specificity is directed toward compounds with electrophilic sites.ligandin is a cytoplasmic protein found in abundance in the liver of rat, man, and other species. This protein is capable of binding noncovalently a large number of compounds, which includes bilirubin, heme, benzyl penicillin, certain steroids, and such dyes as bromosulfophthalein and indocyanine green (1, 2). Phylogenetic, ontogenetic, induction, and competition studies support the hypothesis that ligandin is a major determinant of the net flux of organic anions from plasma into the liver (3-7). Fractionation of the protein on the basis of any one of its binding activities has resulted in apparently identical, highly purified preparations (7-9); Thus, the term, ligandin (2), is synonymous with that of azo-dye carcinogenbinding protein (8), corticosteroid binding I protein (9), and Y protein (7)$, §.The glutathione S-transferases (EC 2.5.1.18) from rat liver (iO-i3) have sevieral physical properties in common with rat liver ligandin (2). When crude liver extracts were subjected to filtration on Sephadex G-75, the fraction containing ligandin also served as a source of enzymatic activity for the conjugation of glutathione (GSH) with 1,2-dichloro-4-nitrobenzene (14). However, subsequent fractionation resulted in removal of alriost all GSH transferase activity with this substrate despite virtually complete recovery of ligandin (t, 15).Since four of the GSH transferases of rat liver (transferases A, B; C, and E) have been purified to homogeneity (11-13), (w/v) and the precipitate was removed by centrifugation after an additional 4 hr at 4°. The superratant fluid was used directly for enzyme assays with 1,2-ehloro-4-nitrobenzene. The precipitate was washed twice with phosphate-buffered saline at pH 7.4, containing 2%o polyethylene glycol. The residue was dissolved in 0.5 M NaOH, and absorbance at 280 nm was determined.

Vertebrate and invertebrate carotenoid-binding proteins

“…It also possesses other unrelated activities including the ability to catalyze double-bond shift reactions in steroid biosynthesis, cis-trans isomerization of retinoic acid, and noncatalytic binding of various hydrophobic molecules "ligandin" activity [77] although carotenoid binding activity had not been described previously [78][79] We therefore used recombinant human GSTP1 to assess its binding properties with xanthophyll carotenoids. We found that GSTP1 binds dietary zeaxanthin and ocular meso-zeaxanthin with high affinity and specificity, while lutein does not bind.…”

A virus-directed enzyme prodrug therapy (VDEPT) strategy for lung cancer using a CYP2B6/NADPH-cytochrome P450 reductase fusion protein

“…35 The enzyme assay is based the GST-mediated catalysis of the conjugation between Glutathione (GSH) and CDNB (1-chloro-2,4 dinitrobenzene) forming a dinitrophenyl thioether chromophore. CDNB is routinely used as the substrate permitting the detection of most GST isoforms.…”

Glutathione S-Transferase Pi Has at Least Three Distinguishable Xenobiotic Substrate Sites Close to Its Glutathione-binding Site

“…Enzyme Assays-Enzymatic activity toward CDNB was measured in a total volume of 1.0 ml using a Hewlett-Packard 8453 spectrophotometer by monitoring the formation of the conjugate of CDNB (3 mM) and glutathione (2.5 mM) at 340 nm (⌬⑀ ϭ 9600 M Ϫ1 cm Ϫ1 ) in 0.1 M potassium phosphate buffer (pH 6.5) at 25°C, according to the method of Habig et al (32). All measurements were corrected for the spontaneous nonenzymatic rate of formation of the conjugate of glutathione and CDNB.…”