Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of the Catalytic Activity of Glutathione Transferases

A cDNA clone, lambda GTRA8, encoding rat glutathione transferase subunit 8 has been isolated from a lambda gt10 rat hepatoma cDNA library. The previously known amino acid sequence of the enzyme was used to design primers for a polymerase chain reaction that yielded a 0.3 kb DNA fragment from the hepatoma library. The 0.3 kb fragment was used as a probe for screening and a 0.9 kb cDNA clone containing a complete open reading frame was obtained. After DNA sequencing and subcloning into an expression vector, the enzyme was expressed in Escherichia coli and purified. Specific activities and kcat./Km values were determined for a number of substrates, including alpha, beta-unsaturated carbonyl compounds. The highest activity was obtained with 4-hydroxyalkenals and with acrolein, genotoxic products of lipid peroxidation. In addition, the rat class Alpha glutathione transferase 8-8 displays high catalytic activity in the reaction between glutathione and the diuretic drug ethacrynic acid, a compound normally considered as a substrate characteristic for class Pi glutathione transferases.

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Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of Substrate Binding to the Active Site of Glutathione Transferases

Journal of Biological Chemistry ◽

10.1074/jbc.m002819200 ◽

2000 ◽

Vol 276 (8) ◽

pp. 5427-5431 ◽

Cited By ~ 19

Author(s):

Anna Maria Caccuri ◽

Giovanni Antonini ◽

Philip G. Board ◽

Jack Flanagan ◽

Michael W. Parker ◽

...

Keyword(s):

Active Site ◽

Glutathione Transferase ◽

Substrate Binding ◽

Glutathione Transferases ◽

Evolutionary Strategies

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Porcine glutathione transferase Alpha 2-2 is a human GST A3-3 analogue that catalyses steroid double-bond isomerization

Biochemical Journal ◽

10.1042/bj20100839 ◽

2010 ◽

Vol 431 (1) ◽

pp. 159-167 ◽

Cited By ~ 13

Author(s):

Natalia Fedulova ◽

Françoise Raffalli-Mathieu ◽

Bengt Mannervik

Keyword(s):

Double Bond ◽

Glutathione Transferase ◽

Biological Species ◽

Glutathione Transferases ◽

Special Role ◽

Primary Role ◽

The Novel ◽

Protective Function ◽

Isomerase Activity

A primary role of GSTs (glutathione transferases) is detoxication of electrophilic compounds. In addition to this protective function, hGST (human GST) A3-3, a member of the Alpha class of soluble GSTs, has prominent steroid double-bond isomerase activity. The isomerase reaction is an obligatory step in the biosynthesis of steroid hormones, indicating a special role of hGST A3-3 in steroidogenic tissues. An analogous GST with high steroid isomerase activity has so far not been found in any other biological species. In the present study, we characterized a Sus scrofa (pig) enzyme, pGST A2-2, displaying high steroid isomerase activity. High levels of pGST A2-2 expression were found in ovary, testis and liver. In its functional properties, other than steroid isomerization, pGST A2-2 was most similar to hGST A3-3. The properties of the novel porcine enzyme lend support to the notion that particular GSTs play an important role in steroidogenesis.

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Mitochondrial Glutathione Transferases. The Alkylation of Mitochondrial Membrane Yields a Catalyst with Glutathione-Transferase-Like Properties

Biological Chemistry Hoppe-Seyler ◽

10.1515/bchm3.1986.367.2.937 ◽

1986 ◽

Vol 367 (2) ◽

pp. 937-942 ◽

Cited By ~ 4

Author(s):

Pavel KRAUS ◽

Jutta WIGAND ◽

Ralf OSTERMAIER

Keyword(s):

Mitochondrial Membrane ◽

Glutathione Transferase ◽

Glutathione Transferases ◽

Mitochondrial Glutathione

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Alteration in the status of glutathione transferase of the water snail, Bulinus globosus, during aestivation and recovery

Animal Biology ◽

10.1163/157075610x491680 ◽

2010 ◽

Vol 60 (2) ◽

pp. 145-155 ◽

Cited By ~ 1

Author(s):

Yetunde Adedolapo Ojopagogo ◽

Isaac Olusanjo Adewale

Keyword(s):

Glutathione Transferase ◽

Specific Activity ◽

Glutathione Transferases ◽

Partial Purification ◽

Major Protein ◽

High Concentration ◽

Cibacron Blue ◽

The Status ◽

Bulinus Globosus ◽

Specific Activities

AbstractThe varying status of glutathione transferases (GSTs) in water snail, Bulinus globosus, an intermediate host of disease-causing Schistosoma haematobium (Bilharz 1852) has been investigated. The expression of GST isoenzymes in the water snail appears seasonal with about three isoenzymes appearing during raining season, when the organism is active, which may reduce to a single peak of one isoenzyme during aestivation, when the organism is inactive. GST isoenzyme is present in high concentration in all the tissues investigated namely: haemolymph, foot muscle and hepatopancreas with specific activities of 0.006 ± 0.002, 0.45 ± 0.021 and 1.33 ± 0.103 units/mg protein respectively for 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. With this substrate, the specific activity of GST from the hepatopancreas appears higher than the specific activities that have been previously reported for GSTs from molluscs. Partial purification of the isoenzymes using Tris acrylic acid-based resins enabled us to observe that GST appears to be the major protein in the hepatopancreas of this organism. We also found indications for the presence of an endogenous GST inhibitor in the cytosol, whose function is yet unknown. All the traditional GST inhibitors such as cibacron blue, hematin, bromosulfophthalein and S-hexylglutathione were able to inhibit the isoenzymes effectively, with cibacron blue being the most potent. The isoenzymes however have narrow substrate specificity. We conclude that different isoenzymes of GST are expressed in the same class of molluscs, even when they belong to the same genus or species, and that the expression may depend on whether the snails are on aestivation or not.

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The potency of inducers of NAD(P)H:(quinone-acceptor) oxidoreductase parallels their efficiency as substrates for glutathione transferases. Structural and electronic correlations

Biochemical Journal ◽

10.1042/bj2730711 ◽

1991 ◽

Vol 273 (3) ◽

pp. 711-717 ◽

Cited By ~ 66

Author(s):

S R Spencer ◽

L A Xue ◽

E M Klenz ◽

P Talalay

Keyword(s):

Molecular Mechanisms ◽

Glutathione Transferase ◽

Quinone Reductase ◽

Detoxification Enzymes ◽

Glutathione Transferases ◽

Methyl Cinnamate ◽

Major Mechanism ◽

Electronic Densities ◽

Quinone Acceptor ◽

Inductive Activity

Induction of glutathione transferases (EC. 2.5.1.18), NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2; quinone reductase) and other detoxification enzymes is a major mechanism for protecting cells against the toxicities of electrophiles, including many carcinogens. Although inducers of these two enzymes belong to many different chemical classes, they nevertheless contain (or acquire by metabolism) electrophilic centres that appear to be essential for inclusive activity, and many inducers are Michael reaction acceptors [Talalay, De Long & Prochaska (1988) Proc. Natl. Acad. Sci. U.S.A., 85, 8261-8265]. The inducers therefore share structural and electronic features with glutathione transferase substrates. To define these features more precisely, we examined the inductive potencies (by measuring quinone reductase in murine hepatoma cells) of two types of glutathione transferase substrates: a series of 1-chloro-2-nitrobenzenes bearing para-oriented electron-donating or -withdrawing substituents and a wide variety of other commonly used and structurally unrelated glutathione transferase substrates. We conclude that virtually all glutathione transferase substrates are inducers, and their potencies in the nitrobenzene series correlate linearly with the Hammett sigma or sigma- values of the aromatic substituents, precisely as previously reported for their efficiencies as glutathione transferase substrates. More detailed information on the electronic requirements for inductive activity was obtained with a series of methyl trans-cinnamates bearing electron-withdrawing or -donating substituents on the aromatic ring, and in which the electronic densities at the olefinic and adjacent carbon atoms were measured by 13C n.m.r. Electron-withdrawing meta-substituents markedly enhance inductive potency in parallel with their increased non-enzymic reactivity with GSH. Thus, methyl 3-bromo-, 3-nitro- and 3-chloro-cinnamates are 21, 14 and 8 times more potent inducers than the parent methyl cinnamate. This finding permits the design of more potent inducers, which are important for elucidation of the molecular mechanisms of induction.

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Leukotriene C synthase in mouse mastocytoma cells. An enzyme distinct from cytosolic and microsomal glutathione transferases

Biochemical Journal ◽

10.1042/bj2500713 ◽

1988 ◽

Vol 250 (3) ◽

pp. 713-718 ◽

Cited By ~ 37

Author(s):

M Söderström ◽

S Hammarström ◽

B Mannervik

Keyword(s):

Microsomal Fraction ◽

Glutathione Transferase ◽

Specific Activity ◽

Glutathione Transferases ◽

Leukotriene C4 ◽

Cytosol Fraction ◽

Mastocytoma Cells ◽

Specific Activities ◽

Leukotriene A4 ◽

Microsomal Glutathione

Leukotriene C4 synthesis was studied in preparations from mouse mastocytoma cells. Enzymic conjugation of leukotriene A4 with glutathione was catalysed by both the cytosol and the microsomal fraction. The specific activity of the microsomal fraction (7.8 nmol/min per mg of protein) was 17 times that of the cytosol fraction. The cytosol fraction of the mastocytoma cells contained two glutathione transferases, which were purified to homogeneity and characterized. A microsomal glutathione transferase was purified from mouse liver; this enzyme was shown by immunoblot analysis to be present in the mastocytoma microsomal fraction at a concentration one-tenth or less of that in the liver microsomal fraction. Both the cytosolic and the microsomal glutathione transferases in the mastocytoma cells were identified with enzymes previously characterized, by determining specific activities with various substrates, sensitivities to inhibitors, reactions with antibodies, and physical properties. The purified microsomal glutathione transferase from liver was inactive with leukotriene A4 or its methyl ester as substrate. The cytosolic enzymes displayed activity with leukotriene A4, but their specific activities and intracellular concentrations were too low to account for the leukotriene C4 formation in the mastocytoma cells. The microsomal fraction of the cells contained an enzyme distinguishable by various criteria from the previously studied glutathione transferases. This membrane-bound enzyme, leukotriene C synthase (leukotriene A4:glutathione S-leukotrienyltransferase), appears to carry the main responsibility for the biosynthesis of leukotriene C4.

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Structural Basis of the Suppressed Catalytic Activity of Wild-type Human Glutathione Transferase T1-1 Compared to its W234R Mutant

Journal of Molecular Biology ◽

10.1016/j.jmb.2005.10.049 ◽

2006 ◽

Vol 355 (1) ◽

pp. 96-105 ◽

Cited By ~ 29

Author(s):

Kaspars Tars ◽

Anna-Karin Larsson ◽

Abeer Shokeer ◽

Birgit Olin ◽

Bengt Mannervik ◽

...

Keyword(s):

Catalytic Activity ◽

Glutathione Transferase ◽

Structural Basis ◽

Wild Type

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A Peroxisomal Glutathione Transferase of Saccharomyces cerevisiae Is Functionally Related to Sulfur Amino Acid Metabolism

Eukaryotic Cell ◽

10.1128/ec.00216-06 ◽

2006 ◽

Vol 5 (10) ◽

pp. 1748-1759 ◽

Cited By ~ 28

Author(s):

Lina Barreto ◽

Ana Garcerá ◽

Kristina Jansson ◽

Per Sunnerhagen ◽

Enrique Herrero

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Amino Acid Metabolism ◽

Redox Regulation ◽

Acid Metabolism ◽

Glutathione Transferase ◽

Reduced Glutathione ◽

Glutathione Transferases ◽

Sulfur Amino Acid ◽

Altered Expression

ABSTRACT Saccharomyces cerevisiae cells contain three omega-class glutathione transferases with glutaredoxin activity (Gto1, Gto2, and Gto3), in addition to two glutathione transferases (Gtt1 and Gtt2) not classifiable into standard classes. Gto1 is located at the peroxisomes, where it is targeted through a PTS1-type sequence, whereas Gto2 and Gto3 are in the cytosol. Among the GTO genes, GTO2 shows the strongest induction of expression by agents such as diamide, 1-chloro-2,4-dinitrobenzene, tert-butyl hydroperoxide or cadmium, in a manner that is dependent on transcriptional factors Yap1 and/or Msn2/4. Diamide and 1-chloro-2,4-dinitrobenzene (causing depletion of reduced glutathione) also induce expression of GTO1 over basal levels. Phenotypic analyses with single and multiple mutants in the S. cerevisiae glutathione transferase genes show that, in the absence of Gto1 and the two Gtt proteins, cells display increased sensitivity to cadmium. A gto1-null mutant also shows growth defects on oleic acid-based medium, which is indicative of abnormal peroxisomal functions, and altered expression of genes related to sulfur amino acid metabolism. As a consequence, growth of the gto1 mutant is delayed in growth medium without lysine, serine, or threonine, and the mutant cells have low levels of reduced glutathione. The role of Gto1 at the S. cerevisiae peroxisomes could be related to the redox regulation of the Str3 cystathionine β-lyase protein. This protein is also located at the peroxisomes in S. cerevisiae, where it is involved in transulfuration of cysteine into homocysteine, and requires a conserved cysteine residue for its biological activity.

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Blocking factors and the isolation of glutathione transferases from Hymenolepis diminuta (Cestoda: Cyclophyllidea)

Parasitology ◽

10.1017/s0031182000060212 ◽

1990 ◽

Vol 100 (1) ◽

pp. 137-141 ◽

Cited By ~ 7

Author(s):

P. M. Brophy ◽

J. Barrett

Keyword(s):

Catalytic Activity ◽

Hymenolepis Diminuta ◽

Glutathione Transferases ◽

Total Soluble Protein ◽

Partial Purification ◽

Transferase Activity ◽

Affinity Matrix ◽

Blocking Factors ◽

And Function ◽

Subunit Size

SummaryFour acidic glutathione (GSH) transferase forms were isolated from the cytosol of the adult cestode Hymenolepis diminuta by hydroxylapatite chromatography, glutathione-affinity chromatography and chromatofocusing, pH 7–5. The enzymes were dimers of subunit size approximately 24 kDa and accounted for at least 3% of the total soluble protein. The major GSH transferase had limited catalytic activity but may interact with a range of ligands and function as a binding/passive detoxification protein. An endogenous factor interfered with the binding of the crude cytosolic GSH transferase activity to glutathione-dependent affinity matrices but, following partial purification, the GSH transferase activity successfully interacted with the glutathione affinity matrix.

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