Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A study of the structure of glutathione transferase B (ligandin) has been made with a view to understanding the relationship between the structures of the subunits of which it is composed. It consists of a mixture of a homodimer (YaYa) and a heterodimer (YaYc) in which the monomers are defined by their apparent molecular weights, that of Ya being 22000 and Yc 25000. Soluble tryptic peptides from the native homodimer YaYa have been compared with those from an artificial homodimer YcYc produced by rehybridization of native YaYc. Approximately 10 peptides specific to YaYa, 12 specific to YcYc and 21 common to both have been detected. Some of the above peptides are derived from variants of the monomers themselves. YaYa and YcYc have two C termini which are the same in both dimers, namely phenylalanine and lysine. Also there are four cysteinyl peptides, of which three are common to YaYa and YcYc and one specific to each. These results suggest that Ya and Yc are derived from at least two different but related genes.
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PMID:Evidence that the Ya and Yc subunits of glutathione transferase B (ligandin) are the products of separate genes. 714 Jul 37

After the intraperitoneal administration of radioactive cholic acid to rats, the binding of the cholic acid to protein was immunologically investigated. The serum and liver cytosol were separately incubated with antiligandin or antialbumin immunoglobulin and then subjected to sucrose density gradient centrifugation. The sedimentation behavior of the radioactivity, endogenous bile acids, glutathione S-transferase activity of ligandin and protein provided evidence that cholic acid is bound to both ligandin and albumin in liver, and mostly to albumin in serum. However, little, if any, cholic acid seems to be bound to protein in bile. These results suggest that ligandin and albumin are the major physiological carriers of bile acid.
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PMID:The binding of cholic acid to protein in rat serum and liver. 733 10

The number of binding sites and the dissociation constants were determined for the binding of bilirubin to human liver ligandin and to human serum albumin. Albumin has a primary bilirubin binding site (KD = 0.03 microM), measured by the peroxidase procedure, and two apparently equivalent secondary binding sites (KD = 2 microM), determined by fluorescence quenching experiments. By contrast, ligandin does not have a corresponding high affinity site. The absence of this high affinity site was shown both by the peroxidase procedure and by direct competition between albumin and ligandin for bilirubin. Bilirubin binding to ligandin, measured by fluorescence quenching, is complex. At both pH 6.5 and 7.4, two interacting sites were observed with a Hill coefficient of 1.5, K' approximately 5 microM. Bilirubin binding to ligandin is not independent of glutathione S-transferase activity. Depending upon pH and upon the order with which the reactants are added, bilirubin can markedly alter the transferase activity. The results are interpreted in terms of kinetically stable conformational isomers of ligandin induced by bilirubin or by glutathione.
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PMID:Bilirubin binding to human liver ligandin (glutathione S-transferase). 737 7

Previously, we identified a soluble protein from Hyoscyamus muticus that was photolabeled by 5-azido-indole-3-acetic acid. This protein was determined to be a glutathione S-transferase (GST; J. Bilang, H. Macdonald, P.J. King, and A. Sturm [1993] Plant Physiol 102: 29-34). We have examined the effect of auxin on the activity of this H. muticus GST. Auxins reduced enzyme activity only at high concentrations, with 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid being more effective than indole-3-acetic acid (IAA) and naphthylacetic acid. IAA was a noncompetitive inhibitor, whereas inhibition by 2,4-D was competitive with respect to 1-chloro-2,4-dinitro-benzene. We also present the sequence of a full-length cDNA clone that codes for a GST and contains all partial amino acid sequences of the purified protein. The auxin-binding GST was found in high amounts in roots and stems and low amounts in leaves and flower buds. The steady-state mRNA level was not regulated by IAA or naphthylacetic acid, whereas 2,4-D and 2,3-dichlorophenoxyacetic acid increased mRNA levels. We propose a model in which 2,4-D is a substrate for GST, whereas IAA binds at a second site, known as a ligandin-binding site for the purpose of intracellular transport.
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PMID:Cloning and characterization of a glutathione S-transferase that can be photolabeled with 5-azido-indole-3-acetic acid. 748 Mar 25

Organic anions can be excreted from the liver into the bile or back into the general circulation (sinusoidal efflux). It has previously been shown that the net sinusoidal efflux rate of dibromosulfophthalein from the perfused liver into the perfusate is the result of actual efflux from and reuptake into the liver, and can be strongly influenced by the presence of bovine serum albumin in the perfusion medium. The present study investigated whether the influence of albumin on the net sinusoidal efflux process is albumin-specific or whether other binding proteins could have a similar effect on the sinusoidal efflux. Using a single-pass liver perfusion technique and short-lasting (pulse) protein infusions, the stimulatory effect of a wide range of dibromosulfophthalein binding proteins on the sinusoidal efflux process were determined. These experiments showed that all the serum albumins tested as well as the liver cytosolic binding proteins fatty acid binding protein and ligandin (glutathione S-transferase) stimulated this process. The other proteins tested, bovine beta lactoglobulin-b, human gamma globulin and chicken egg lysozyme showed no stimulatory effect, despite relatively high equilibrium binding of dibromosulfophthalein. No clear-cut relationship was found between the equilibrium unbound ligand concentration as measured in perfusate and the stimulatory effect, suggesting absence of equilibrium binding in the sinusoids. Equilibrium binding of dibromosulfophthalein to chicken serum albumin and ligandin as well as the dissociation rate constants were determined in vitro with rapid filtration techniques.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The sinusoidal efflux of dibromosulfophthalein from rat liver is stimulated by albumin, ligandin and fatty acid binding protein but not by other dibromosulfophthalein binding proteins. 752 96

To examine the hypothesis that glutathione S-transferases (GST) play an important role in the hepatocellular transport of hydrophobic organic anions, the kinetics of the spontaneous transfer of unconjugated bilirubin between membrane vesicles and rat liver glutathione S-transferase B (ligandin) was studied, using stopped-flow fluorometry. Bilirubin transfer from glutathione S-transferase B to phosphatidylcholine vesicles was best described by a single exponential function, with a rate constant of 8.0 +/- 0.7 s-1 (+/- SD) at 25 degrees C. The variations in transfer rate with respect to acceptor phospholipid concentration provide strong evidence for aqueous diffusion of free bilirubin. This finding was verified using rhodamine-labeled microsomal membranes as acceptors. Bilirubin transfer from phospholipid vesicles to GST also exhibited diffusional kinetics. Thermodynamic parameters for bilirubin dissociation from GST were similar to those for human serum albumin. The rate of bilirubin transfer from rat liver basolateral plasma membranes to acceptor vesicles in the presence of glutathione S-transferase B declined asymptotically with increasing GST concentration. These data suggest that glutathione S-transferase B does not function as an intracellular bilirubin transporter, although expression of this protein may serve to regulate the delivery of bilirubin, and other nonsubstrate ligands, to sites of metabolism within the cell.
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PMID:Influence of glutathione S-transferase B (ligandin) on the intermembrane transfer of bilirubin. Implications for the intracellular transport of nonsubstrate ligands in hepatocytes. 756 84

Human glutathione transferase A1-1 can be expressed as a fusion protein with coat protein III of filamentous phage f1 in a form that allows selection among variant mutant forms based on specific adsorption to immobilized active-site ligands. A library of mutant enzymes differing in the active-site region was generated by random mutagenesis of ten amino acid residues involved in the binding of electrophilic substrates. Novel glutathione transferases with altered specificity for active-site ligands were isolated by adsorption of the fusion protein on the surface of phage to analogs of an electrophilic substrate. Thus, phage display of glutathione transferase affords a system for engineering novel binding specificities onto the pre-existing protein framework of the enzyme.
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PMID:Glutathione transferases with novel active sites isolated by phage display from a library of random mutants. 760 63

Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase A1-1 have shown that N epsilon of this amino acid residue is adjacent to the sulfur atom of a glutathione derivative bound to the active site, suggesting the presence of a hydrogen bond. The phenolic hydroxyl group of Tyr9 also forms a hydrogen bond to the sulfur atom of glutathione, and removal of this hydroxyl group causes partial inactivation of the enzyme. The present study demonstrates by use of site-directed mutagenesis the functional significance of Arg15 for catalysis. Mutation of Arg15 into Leu reduced the catalytic activity by 25-fold, whereas substitution by Lys caused only a threefold decrease, indicating the significance of a positively charged residue at position 15. Mutation of Arg15 into Ala or His caused a substantial reduction of the specific activity (200 or 400-fold, respectively), one order of magnitude more pronounced than the effect of the Tyr9-->Phe mutation. Double mutations involving residues 9 and 15 demonstrated that the effects of mutations at the two positions were additive except for the substitution of His for Arg15, which appeared to cause secondary structural effects. The pKa value of the phenolic hydroxyl of Tyr9 was determined by UV absorption difference spectroscopy and was found to be 8.1 in the wild-type enzyme. The corresponding pKa values of mutants R15K, R15H and R15L were 8.5, 8.7 and 8.8, respectively, demonstrating the contribution of the guanidinium group of Arg15 to the electrostatic field in the active site. Addition of glutathione caused an increased pKa value of Tyr9; this effect was not obtained with S-methylglutathione. These results show that Tyr9 is protonated when glutathione is bound to the enzyme at physiological pH values. The involvement of an Arg residue in the binding and activation of glutathione is a feature that distinguishes class Alpha glutathione transferases from members in other glutathione transferase classes.
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PMID:Functional significance of arginine 15 in the active site of human class alpha glutathione transferase A1-1. 772 30

Alkylating agents can be detoxified by conjugation with glutathione (GSH). One of the physiological significances of this lies in the observation that cancer cells resistant to the cytotoxic effects of alkylating agents have higher levels of GSH and high glutathione S-transferase (GST) activity. However, little is known about the GSH-/GST-dependent biotransformation of alkylating agents, including cyclophosphamide. Cyclophosphamide becomes cytostatic after the enzymatic formation of 4-hydroxycyclophosphamide. The ultimate alkylating species formed from cyclophosphamide is phosphoramide mustard. In this paper we describe the involvement of purified human glutathione S-transferases isoenzymes GST A1-1, A2-2, M1a-1a, and P1-1 in the formation of two types of glutathionyl conjugates of cyclophosphamide, i.e., 4-glutathionylcyclophosphamide (4-GSCP) and monochloromonoglutathionylphosphoramide mustard. When 0.1 mM 4-hydroxycyclophosphamide and 1 mM GSH was incubated in the presence of 10 microM GST A1-1, A2-2, M1a-1a, and P1-1 the formation of 4-GSCP was 2-4-fold increased above the spontaneous level. Enzyme kinetic analysis demonstrated the lowest Km (0.35 mM) for GST A1-1. Km values for the other GST enzymes ranged from 1.0 to 1.9 mM. Glutathione S-transferase A1-1 (40 microM) also increased the conjugation of phosphoramide mustard and GSH (both 1 mM) 2-fold, while the other major human isoenzymes, A2-2, M1a-1a, and P1-1, did not influence the formation of monochloromonoglutathionylphosphoramide mustard. These results indicate that only one enzyme within the class of human GST alpha enzymes was able to catalyze the reaction of the aziridinium ion of phosphoramide mustard with glutathione. Thus increased levels of GST A1-1 in tumor cells can contribute to an enhanced detoxification of phosphoramide mustard and hence to the development of drug resistance. Since all of the human GSTs tested did catalyze the formation of 4-GSCP, the role of 4-GSCP either as a transport form of activated cyclophosphamide or as a detoxification product is discussed.
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PMID:Involvement of human glutathione S-transferase isoenzymes in the conjugation of cyclophosphamide metabolites with glutathione. 795 69

Human glutathione transferase A1-1 (GST A1-1) is a detoxifying enzyme catalyzing the conjugation of glutathione with a variety of hydrophobic, electrophilic substrates. When the role of the hydrophobic substrate-binding site residue Met208 was investigated by random mutagenesis, introduction of charged amino acid residues had the greatest deleterious effect on enzyme activity. However, in the lysine mutant some of the lost activity could be regained by the addition of a benzoic acid derivative to the reaction mixture. The activating molecule has now been optimized such that all activity is recovered. The most potent activator, 4-propylbenzoic acid, has been used in studies of the mechanism behind the activation. A heterodimeric species of GST A1-1, containing only one activatable subunit, has been constructed. The heterodimer shows a strictly additive activation curve when compared to its parental forms, indicating that the activation is not due to co-operativity between the subunits. Furthermore, a novel electrophilic substrate, 4-chloro-3,5-dinitrobenzoic acid, with a carboxylate group expected to interact with residue 208 gives a higher kcat value with the lysine mutant than with wild-type GST A1-1. All results obtained in the here support the view that the positive charge introduced into the lysine mutant adversely affects the structure of the C-terminal helix of this enzyme, preventing it from adopting the conformation needed for full activity. The negatively charged carboxylate group of the activator probably neutralizes the positive charge of the side-chain amino group and thereby restores the substrate-binding site to a form that is favorable for the catalytic function.
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PMID:Benzoic acid derivatives induce recovery of catalytic activity in the partially inactive Met208Lys mutant of human glutathione transferase A1-1. 1032 79


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