The glutathione S-transferases (GST) represent a major group of detoxification enzymes. All eukaryotic species possess multiple cytosolic and membrane-bound GST isoenzymes, each of which displays distinct catalytic as well as noncatalytic binding properties: the cytosolic enzymes are encoded by at least five distantly related gene families (designated class alpha, mu, pi, sigma, and theta GST), whereas the membrane-bound enzymes, microsomal GST and leukotriene C, synthetase, are encoded by single genes and both have arisen separately from the soluble GST. Evidence suggests that the level of expression of GST is a crucial factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals. In this article the biochemical functions of GST are described to show how individual isoenzymes contribute to resistance to carcinogens, antitumor drugs, environmental pollutants, and products of oxidative stress.A description of the mechanisms of transcriptional and posttranscriptional regulat...

The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance.

The glutathione transferases are recognized as important catalysts in the biotransformation of xenobiotics, including drugs as well as environmental pollutants. Multiple forms exist, and numerous transferases from mammalian tissues, insects, and plants have been isolated and characterized. Enzymatic properties, reactions with antibodies, and structural characteristics have been used for classification of the glutathione transferases. The cytosolic mammalian enzymes could be grouped into three distinct classes--Alpha, Mu, and Pi; the microsomal glutathione transferase differs greatly from all the cytosolic enzymes. Members of each enzyme class have been identified in human, rat, and mouse tissues. Comparison of known primary structures of representatives of each class suggests a divergent evolution of the enzyme proteins from a common precursor. Products of oxidative metabolism such as organic hydroperoxides, epoxides, quinones, and activated alkenes are possible "natural" substrates for the glutathione transferases. Particularly noteworthy are 4-hydroxyalkenals, which are among the best substrates found. Homologous series of substrates give information about the properties of the corresponding binding site. The catalytic mechanism and the active-site topology have been probed also by use of chiral substrates. Steady-state kinetics have provided evidence for a "sequential" mechanism.

Glutathione transferases--structure and catalytic activity.

Membrane lipid composition and cellular function.

Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase.

Publisher Summary Glutathione (GSH) is the most important nonprotein thiol in living systems and is of widespread occurrence in the intracellular milieu of animals, plants, and microorganisms GSH was isolated and named by the English biochemist Frederick Gowland Hopkins This chapter discusses GSH status, the biologically relevant chemistry of GSH, the forms in which GSH can be present within the cell, along with the GSH content of cells and the methods for analysis of this substance GSH-related biochemical reactions and the biological roles of GSH are discussed in the chapter The use of perturbations in GSH status as a means for investigating GSH-related phenomena and an analysis of the consequences of perturbation are presented A short summary of genetic lesions related to GSH is also included Like chemically induced perturbations in GSH status, genetic lesions provide valuable insights into the role of GSH in normal functions and processes in cells The chapter concludes with some brief comments about the future of the relationship of GSH status to cellular processes

The Glutathione Status of Cells

Publisher Summary This chapter provides the spectrophotometric, titrimetric, nitrite, and cyanide assay for the differentiation of glutathione S-transferases. Spectrophotometric assays depend upon a direct change in the absorbance of the substrate when it is conjugated with glutathione (GSH). Because each of the reactions is catalyzed at a finite rate in the absence of enzyme, care is needed to reduce nonenzymatic catalysis by minimizing substrate concentrations and by decreasing pH wherever necessary. Titrimetric assay is based on the principle that the conjugation of alkyl halides with GSH can be measured titrimetrically. Although acid production accompanies many of the transferase catalyzed reactions in which thioethers are formed, titrimetry is only used when more convenient assays are not available. Nitrite assay is based on the principle that nitrite is released when GSH reacts with nitroalkanes or with organic nitrate esters. The nitrite can be assayed as the limiting factor in a diazotization reaction with sulfanilamide that produces a readily quantitatable pink dye. Cyanide assay is based on the fact that when glutathione transferases catalyze the attack of the glutathione thiolate ion on the electrophilic sulfur atom of several organic thiocyanates, it results in the formation of an asymmetric glutathionyl disulfide and cyanide. Cyanide can be readily quantitated by a calorimetric method.

Assays for differentiation of glutathione S-transferases.

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 produced against ligandin, bear entirely similar 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 shown to competitively inhibit the conjugation reaction. It is suggested that specificity is directed toward compounds with electrophilic sites.

https://www.pnas.org/content/pnas/71/10/3879.full.pdf

The identity of glutathione S-transferase B with ligandin, a major binding protein of liver.

Publisher Summary This chapter presents a procedure for the preparation of glutathione transferases of the rat and the human. The glutathione S-transferases are the enzymes catalyzing conjugation reactions with glutathione as the first step in mercapturic acid synthesis. Although these enzymes may be distinguished from each other by their characteristic substrate-activity spectrum; all of the transferases are active with 1-chloro-2,4-dinitrobenzene as substrate. The reaction of this substrate and glutathione occurs spontaneously and the assay, therefore, requires the use of a control from which enzyme is absent. Not all of the data presented in the chapter summarizing the purification procedure were obtained with 1-chloro-2,4-dinitrobenzene. Methyl iodide, 1,2-dichloro-4-nitrobenzene, and 1,2-epoxy-3-(p-nitrophenoxy)propane have also been used. The homogeneous transferases prepared from human and rat liver represent a group that, aside from overlapping substrate specificity, resemble each other in size and subunit number. Although transferases A and C are similar to a greater degree, by reason of cross-reactivity to antibody raised against either protein, the other transferases from rat liver seem to be quite separate species.

Glutathione S-transferases (rat and human).

Evidence ispresented thatligandin, an intracellular protein involved inthebinding ofsuchanions asbilirubin, indocyanine green, andpenicillin, isidentical toglutathione S-transferase B (EC2.5.1.18), an enzyme catalyzing theconjugation ofglutathione withsuchelec- trophiles as1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4- nitrobenzene, iodomethane, ethacrynic acid,andbromo- sulfophthalein. Theproteins, isolated bydistinct methods, havethesamespecificity forsubstrates andforligands, reactinidentical fashion toantibody pioducedagainst ligandin, bearentirely sinlilar physical characteristics and aminoacidcomposition, andarebothinduced inresponse tophenobarbital. Indocyanine green, oneoftheligands thatisnoteffective asasubstrate, wasshowhtocompeti- tively inhibit theconjugation reaction. Itissuggested that specificity isdirected towardcompoundswithelectrophilic sites. ligandin isacytoplasmic protein found inabundance inthe liver ofrat, man,andother species. Thisprotein iscapable ofbinding noncovalently alarge number ofcompounds, which includes bilirubin, heme,benzyl penicillin, certain steroids, andsuchdyesasbromosulfophthalein

William H. Habig

Papers

Assays for differentiation of glutathione S-transferases.

The identity of glutathione S-transferase B with ligandin, a major binding protein of liver.

Glutathione S-transferases (rat and human).

TheIdentity ofGlutathione S-Transferase B withLigandin, aMajorBinding Protein ofLiver

Glutathione transferase from human erythrocytes. Nonidentity with the enzymes from liver.