William B. Jakoby

[51] Assays for differentiation of glutathione S-Transferases

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.

[27] Glutathione S-transferases (rat and human)

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

Glutathione transferase ϱ has been purified to homogeneity from human erythrocytes. The enzyme has a molecular weight of 47,500 and is composed of two subunits of the same apparent molecular weight. The enzyme is active in catalyzing the reaction of glutathione, as a nucleophile, with a variety of compounds bearing an electrophilic center. Thioether formation with 1-chloro-2,4-dinitrobenzene and with ethacrynic acid are among the most rapid reactions under standard assay conditions. The erythrocyte enzyme has a different amino acid composition from that of glutathione transferases from human liver, whereas all of the liver enzymes appear to have the same composition. The erythrocyte transferase also differs from the liver transferases in its low isoelectric point of 4.5, its lack of reactivity with antibody produced against a liver glutathione transferase, and its more limited spectrum of substrates. The enzyme is present at a concentration of about 1.2 mg/100 ml of packed human erythrocytes.

Glutathione S-transferase AA from rat liver

Glutathione S-transferase AA from rat liver was purified to apparent homogeneity as judged by gel filtration and gel electrofocusing. The protein has an isoelectric point near pH 9.9 and a molecular weight of 45,000 and is composed of two apparently identical subunits. The enzyme is most active with 1-chloro-2,4-dinitrobenzene and glutathione as substrates. The catalytic properties of transferase AA are very similar to those of transferase B although the two proteins differ in their ability to bind bilirubin and other ligands, in their amino acid composition, and in their immunological properties. When mixtures of transferase AA with other purified glutathione S-transferases were denatured in 6 m guanidine hydrochloride and then renatured by dilution, no hybrids were formed as judged by gel electrofocusing. Isolation of the enzymes from a single rat liver revealed the presence of each of the known glutathione transferases.

Aryl sulfotransferase IV from rat liver

Abstract A group of aryl sulfotransferases has been identified that catalyzes sulfate ester formation with simple phenols at an acidic pH and with several physiological metabolites at a more alkaline pH. One enzyme, aryl sulfotransferase IV, has been purified to homogeneity and found to be a protein of 61,000 daltons composed of two subunits of apparent equal size. Homogeneous preparations are active with simple phenols, organic hydroxylamines, and catecholamines as well as serotonin and its metabolites. The enzyme is also active with tyrosine methyl ester and with those peptide hormones e.g., cholecystokinin heptapeptide and some of the enkephalins, which have N-terminal tyrosine residues.

The glutathione S-transferases as a possible detoxification system of rat intestinal epithelium

Abstract Because the glutathione S-transferases perform a detoxification function in liver and kidney, evidence for them was sought in the intestine, another major site of contact with xenobiotics. The range of activity with several different substrates was similar to that of liver: highest activity was observed with 1-chioro-2,4-dinitrobenzene. Antibodies prepared against homogeneous rat liver transfer-ases A, B, and E gave lines of identity with cytosol obtained from intestinal epithelial cells. With 1-chloro-2,4-dinitrobenzene as substrate a shallow gradient of increasing activity was observed from crypt to tip cells of jejunal epithelium. In cells at all stages of maturation, activity was increased in response to phenobarbital whereas alkaline phosphatase, thymidine kinase and γ-glutamyltranspeptidase were not induced. Activity was greatest in the duodenum and jejunum, and lowest in the colon and stomach. The data arc consistent with a detoxification role for the glutathione S-transferases in intestine.

Glutathione S-transferase in the formation of cyanide from organic thiocyanates and as an organic nitrate reductase

Abstract Evidence is presented that glutathione S-transferases, a group of enzymes active in the formation of thioethers from glutathione and a large number of compounds with an electrophilic carbon atom, can also catalyze the formation of nitrous acid and oxidized glutathione from organic nitrate esters such as nitroglycerin. In addition, organic thiocyanates are cleaved by these enzymes to form cyanide and the respective asymmetric disulfide of glutathione.

Thiol S-methyltransferase from rat liver

Abstract The thiol S -methyltransferase from rat liver has been solubilized and prepared in homogeneous form. The enzyme exists in a monomer of M r 28,000 although enzyme activity is highly unstable with a half-life of 4 days under the best conditions of storage. The reaction requires S -adenosylmethionine as methyl donor but, as is the case with many enzymes active in detoxification, a large variety of lipophilic compounds can serve as acceptors. Acceptor activity is limited to thiols. The naturally occurring hydrophilic thiols, glutathione and cysteine, act neither as substrates nor as inhibitors. The course of the reaction is biphasic with an initial rapid formation of product that is followed by a slower linear rate. The suggestion is offered that this behavior reflects the slow dissociation of an enzyme-product complex composed of enzyme and S -adenosyl-homocysteine.

Mercapturic acid formation: The several glutathione transferases of rat liver

Abstract Glutathione transferases catalyze the conjugation of GSH with a number of compounds bearing a nucleophilic group. The number and specificity of such enzymes from rat liver were surveyed. Three enzymes have been purified to homogeneity; each was active with p-nitrobenzyl chloride as substrate. One was also active with epoxides and p-nitrophenethyl bromide; the second with methyl iodide; and the third with 3,4-dichloronitrobenzene and 4-nitropyridine N-oxide. Although there are differences in specificity among the enzyme species, such differences are not directly related to the nucleophilic leaving group. Nor is it accurate to classify the enzymes by their preferences for substrates of a particular type of hydrocarbon skeleton.

Hepatic Accumulation and Intracellular Binding of Conjugated Bilirubin

After the intravenous injection of unconjugated [(3)H]bilirubin into normal Sprague-Dawley and Wistar R rats, radiolabeled bile pigments rapidly accumulated in the liver. By 1.5 min after injection, an average of 36% of the injected isotope was present in liver homogenates. Between 3 and 15 min, 37-64% of the total intrahepatic radiolabeled bilirubin was conjugated, as demonstrated by extraction of label into the polar phase of a solvent partition system. This indicates both rapid conjugation, and accumulation of conjugated bilirubin within the liver cell. Fluorometric determination of the dissociation constants of purified bilirubin and its mono- and diglucuronides for homogeneous preparations of two human and four rat glutathione S-transferases, including ligandin, revealed avid binding of all three bile pigments to this class of proteins. Hence, the observation that the intrahepatic bile pigment pool contains substantial amounts of conjugated bilirubin can be attributed to the high binding affinities observed. Thin-layer chromatographic analysis of the (3)H-pigments produced by p-iodoaniline diazotization of homogenates and cytosol demonstrated that the intrahepatic pool of conjugated bilirubin was almost exclusively monoglucuronide. Examination of radiolabeled bilirubin conjugates excreted in bile during the first 20 min after injection of [(3)H]bilirubin showed no preferential excretion of diglucuronide. These studies indicate that (a) both bilirubin and its monoglucuronide accumulate within the liver cell as ligands with the glutathione S-transferase; and (b) bilirubin diglucuronide does not significantly accumulate within the general intrahepatocellular pool of protein-bound bile pigments. The latter observation is compatible with the formation and excretion of bilirubin diglucuronide directly from the canalicular pool of the liver cell.