Relationship between the soluble glutathione-dependent delta 5-3-ketosteroid isomerase and the glutathione S-transferases of the liver.

Abstract

Soluble, glutathione-stimulated A5-3-ketosteroid isomerase (EC 5.3.3.1) activity of human and rat liver resides in very basic proteins with molecular weights of about 45,000 which are present in high concentrations in these tissues. Physicochemical and immunological evidence is presented for the identity of the proteins responsible for this enzymatic activity with the glutathione S-transferases (RX:glutathione Rtransferase, EC 2.5.1.18) that conjugate glutathione with a variety of electrophilic compounds. In the rat, the steroid isomerase is associated principally with the major transferase (B), which is also known as ligandin, and has the versatility to bind various hydrophobic compounds such as bilirubin, corticosteroids, and metabolites of a number of carcinogens. Other rat liver glutathione S-transferase species are far less active in the steroid isomerization reaction. The A5-3-ketosteroid isomerase activity of human liver is more uniformly distributed among the five glutathione S-transferases that have been described. Steroid isomerization differs fundamentally from other reactions promoted by glutathione S-transferases in that glutathione is not consumed in the reaction. However, because the transferase enzymes promote nucleophilic attack by glutathione on a variety of largely foreign organic substrates, a similar mechanism may be involved in the isomerase reaction. A5-3-Ketosteroids are among the few known naturally occurring substrates for these enzymes.The capacity of various animal and microbial systems to promote the enzymatic conversion of A5-3-ketosteroids (I) to the corresponding a, (3-unsaturated M4-3-ketosteroids (II) was described just over 20 years ago (1, 2). Whereas the enzyme from Pseudomonas testosteroni, a A5-3-ketosteroid isomerase (steroid A-isomerase, 3-oxosteroid A4-A5-isomerase, EC 5.3.3.1) that does not appear to require small molecules for its activity, has been the subject of intensive study from both structural and mechanistic viewpoints (2), far less is known about the corresponding enzymes of animal origin. Recently, Benson and Talalay (3) reported that the cytosol fraction of the livers of a number of species contains A5-3-ketosteroid isomerase activity which is markedly and specifically stimulated by (reduced) glutathione. Partial purification of the rat liver A5-3-ketosteroid isomerase revealed that it was a very basic protein (pI ca. 10) of molecular weight 43,000-45,000 and that it appeared to constitute a substantial fraction of the total proteins of the liver cytosol.These attributes are also characteristic of a family of liver proteins, the glutathione S-transferases which, because of their great versatility of binding and catalytic functions, have been studied in numerous laboratories in the past decade. Indeed, Litwack, Ketterer, and Arias (4) came to the conclusion that the liver proteins which had been investigated independently in each of their laboratories for the ability to bind bilirubin and bromsulfophthalein (5), to bind an azocarcinogen, at least in ...

Isoelectric Focusing of Glutathione S-Transferases From Rat Liver and Kidney

“…175 Transferase B, the predominant transferase in rat liver, is identical with ligandin, previously known as Y-protein, azocarcinogen-binding protein and cortisol-metabolite binder I (Litwack et al, 1971); a role for this protein in the uptake, retention and/or secretion of organic anions by liver and kidney has been proposed (Levi et al, 1969). In addition, transferase B can act as a glutathione peroxidase (Burk et al, 1977;Prohaska & Ganther, 1977) and a 3-oxo A5-steroid isomerase (Benson et al, 1977). Transferases AA, A and C also bind a broad spectrum of non-substrate ligands such as bilirubin and bromosulphophthalein (Ketley et al, 1975).…”

Effects of Oxytetracycline Antibiotic on GST Enzyme Activity and Gene Expression Levels in Rainbow Trout

“…GST catalyzes the conjugation of reduced glutathione (GSH) [18,19]. Additionally several GST isoenzymes exhibit other GSHdependent catalytic activities including the reduction of organic hydroperoxides [20] and isomerization of various unsaturated compounds [21,22,23].…”

Isomerization of the Phytohormone Precursor 12-Oxophytodienoic Acid (OPDA) in the Insect Gut

“…The tautomerization of 2-hydroxymenthofuran to mintlactones constitutes yet another instance of a GST-mediated isomerization reaction; in these, the thiolate form of GSH is proposed to deprotonate the substrate and in this way initiate the reaction (6). The best known example, however, of positional isomerization of double bonds is the conversion of ⌬ 5 -3-ketosteroids to ⌬ 4 -3-ketosteroids, which has been shown to be catalyzed among others by mammalian GSTs (7,8). This case bears the highest formal resemblance to the isomerization of the plant-signaling molecule cis-(ϩ)-12-oxophytodienoic acid, which we found to be catalyzed by insect GSTs (Fig.…”