Affinity labeling of glutathione S-transferase, isozyme 4-4, by 4-(fluorosulfonyl)benzoic acid reveals Tyr115 to be an important determinant of xenobiotic substrate specificity

Barycki, Joseph J.; Colman, Roberta F.

doi:10.1021/bi00211a008

Cited by 43 publications

(29 citation statements)

References 24 publications

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“…The activity toward EA was detected in freshly isolated hepatocytes and did not increase with time and elevated GSTP1 expression, suggesting that other GST family members may also catalyze this reaction. Other reports have also suggested that alpha- (Stenberg et al, 1992), mu- (Barycki and Colman, 1993), and theta- (Hiratsuka et al, 1990) class GSTs contribute to EA conjugation in rat. However, Henderson et al (1998) reported that EA metabolism is lost in the liver of the GSTP knockout mouse.…”

Section: Discussionmentioning

confidence: 87%

Insulin and Glucagon Regulation of GlutathioneS-Transferase Expression in Primary Cultured Rat Hepatocytes

Kim

Woodcroft

Novak

2003

J Pharmacol Exp Ther

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Diabetes is a major cause of morbidity and mortality, and complications resulting from diabetes have been attributed in part to increased oxidative stress. Glutathione S-transferases (GSTs) constitute a major protective mechanism against oxidative stress. Studies of the expression and activity of GSTs during diabetes are inconclusive, with both increased and decreased GST expression being reported in vivo. Insulin and glucagon effects on GST expression and the signaling pathway involved in the glucagon regulation of GST expression were examined in primary cultured rat hepatocytes. The addition of insulin resulted in the elevation of alpha-class GST protein levels, whereas alpha-and pi-class GST protein levels were markedly decreased in hepatocytes cultured with glucagon. In contrast, mu-class GST protein expression was unaffected by insulin or glucagon treatment. Insulin concentrations Ն1 nM resulted in increased GST activities and alpha-class GST protein levels, whereas glucagon concentrations Ն20 nM decreased alpha-and pi-class protein levels and activity. Treatment of cells with 8-bromo-cAMP or dibutyryl-cAMP also resulted in decreased alpha-and pi-class GST protein levels. Pretreatment with N-[2-(4-bromocinnamylamino)ethyl]-5-iso-quinoline sulfonamide (H89), a selective inhibitor of protein kinase A, before glucagon addition markedly attenuated the glucagon effect. This study demonstrates that insulin and glucagon regulate, in an opposing manner, the expression of alpha-class GSTs and that glucagon completely inhibits piclass GST expression in vitro, suggesting that hepatic GST expression may be decreased during diabetes. Furthermore, the present study implicates cAMP and protein kinase A in mediating the inhibitory effect of glucagon on GST expression.Diabetes mellitus is associated with a high risk of atherosclerosis and kidney, nerve, and tissue damage. It has been reported that oxidative stress is increased in diabetic conditions (Traverso et al., 1998) and is a major factor contributing to the extent of chronic diabetes complications (Baynes and Thorpe, 1999). A higher incidence of hepatic cancer has been reported to be associated with diabetes (Adami et al., 1996).The glutathione S-transferases (GSTs), abundantly expressed in liver tissue, constitute one of the major components of the phase II drug-metabolizing enzyme and antioxidant systems. The GSTs catalyze the conjugation of glutathione to a wide range of electrophiles and represent a protective mechanism against oxidative stress (Ketterer, 1998). Several members of the GST family exhibit seleniumindependent glutathione peroxidase activity, which plays an important role in protecting cells against lipid and nucleotide hydroperoxides (Sun et al., 1996;Ketterer, 1998).The GSTs comprise a complex and widespread enzyme superfamily that has been subdivided further into an everincreasing number of classes. The cytosolic GSTs are homo or heterodimeric enzymes, and the major GST subunits expressed in the adult liver are alpha-class subunits A1, A2,...

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Section: Discussionmentioning

confidence: 87%

Insulin and Glucagon Regulation of GlutathioneS-Transferase Expression in Primary Cultured Rat Hepatocytes

Kim

Woodcroft

Novak

2003

J Pharmacol Exp Ther

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“…The enzyme-catalyzed formation of the conjugate between 600 M glutathione and 100 M mBBr was measured in 0.1 M potassium phosphate buffer, pH 6.5, at 25°C using the method of Hulbert and Yakubu (32) with a Perkin-Elmer MPF-3 fluorescence spectrophotometer (excitation at 395 nm and emission at 480 nm). ) (34). Corrections for the nonenzymatic reactions were made for all assays.…”

Section: Materials-frozenmentioning

confidence: 99%

Probing Subunit Interactions in Alpha Class Rat Liver Glutathione S-Transferase with the Photoaffinity Label Glutathionyl S-[4-(Succinimidyl)benzophenone]

Wang

Bauman

Colman

2000

Journal of Biological Chemistry

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Glutathionyl S-[4-(succinimidyl)benzophenone] (GSSucc-BP), an analogue of the product of glutathione and electrophilic substrate, acts as a photoaffinity label of dimeric rat liver glutathione S-transferase (GST), isoenzyme 1-1. A time-dependent loss of enzyme activity is observed upon irradiation of the enzyme with long wavelength UV light in the presence of the reagent. The initial rate of inactivation exhibits nonlinear dependence on the concentration of the reagent, characterized by an apparent dissociation constant of the enzymereagent complex (K R ) of 99 ؎ 2 M and k max of 0.082 ؎ 0.005 min ؊1 . Protection against this inactivation is provided by the electrophilic substrate (ethacrynic acid), electrophilic substrate analogue (dinitrophenol), and product analogues (S-hexylglutathione and p-nitrobenzylglutathione) but not by steroids (⌬ 5 -androstene-3,17-dione and 17␤-estradiol-3,17-disulfate). These results suggest that GS-Succ-BP binds and reacts with the enzyme within the xenobiotic substrate binding site, and this reaction site is distinct from the substrate and nonsubstrate steroid binding sites of the enzyme. About 1 mol of reagent is incorporated into 1 mol of enzyme dimer when the enzyme is completely inactivated. Met-208 is the only amino acid target of the reagent, and modification of this residue in one enzyme subunit of the GST 1-1 dimer completely abolishes the enzyme activity of both subunits. In order to evaluate the role of subunit interactions in the Alpha class glutathione S-transferases, inactive GS-Succ-BP-modified GST 1-1 was mixed with unlabeled, active GST 2-2. The enzyme subunits were dissociated in dilute trifluoroacetic acid and then renatured at pH 7.8 and separated by chromatofocusing into GST 1-1, 1-2, and 2-2. The specific activities of the heterodimer toward several substrates indicate that the loss of catalytic activity in the unmodified subunit of the modified GST 1-1 is the indirect result of the interaction between the two enzyme subunits and that this subunit interaction is absent in the heterodimer GST 1-2.Glutathione S-transferases (GST) 1 (EC 2.5.1.18) are a family of detoxification enzymes that catalyze the conjugation reaction of glutathione with a variety of endogenous electrophiles and xenobiotics (1-4). The enzyme catalyzes the reaction by lowering the pK a of the sulfhydryl group of the enzyme-bound glutathione (5, 6). The conjugation products are more watersoluble, usually less toxic, and can be degraded or transported outside of the cell. Furthermore, GSTs have been implicated in the development of resistance of various tumor cell lines to anti-cancer drugs. A higher level of expression of Alpha class GSTs was observed when Chinese hamster ovary cells were exposed to the nitrogen mustard alkylating agent chlorambucil (7), and cell lines exhibiting overexpression of these GSTs had increased resistance to the anti-cancer drugs chlorambucil and melphalan (8, 9); for recent reviews, see Hayes and Pulford (10) and Van der Aar et al. (11).The mammalian cytosolic GST...

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“…Affinity labeling studies using S-(4-bromo-2,3-dioxobutyl)glutathione and 4-(fluorosulfonyl)benzoic acid demonstrated that Tyr 115 in both isozyme 3-3 and 4 -4 is located in the xenobiotic binding site and contributes to binding of these hydrophobic substrates in addition to its function as a general acid in reactions with certain substrates (i.e. those involving epoxide ring opening and Michael addition reactions) (11,16,17). More recently, Tyr 115 has been identified as a target of other affinity labels (18,19).…”

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confidence: 99%

Monobromobimane as an Affinity Label of the Xenobiotic Binding Site of Rat Glutathione S-Transferase 3-3

Colman

1995

Journal of Biological Chemistry

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Monobromobimane (mBBr), besides being a substrate in the presence of glutathione, inactivates rat liver glutathione S-transferase 3-3 at pH 7.5 and 25°C as assayed using 1-chloro-2,4-dinitrobenzene (CDNB). The rate of inactivation is enhanced about 5-fold by S-methylglutathione. Substrate analogs bromosulfophthalein and 2,4-dinitrophenol decrease the rate of inactivation at least 20-fold. Upon incubation for 60 min with 0.25 mM mBBr and S-methylglutathione, the enzyme loses 91% of its activity toward CDNB and incorporates 2.14 mol of reagent/mol of subunit, whereas incubation under the same conditions but with added protectant 2,4-dinitrophenol yields an enzyme that is catalytically active and contains only 0.89 mol of reagent/mol of subunit. mBBrmodified enzyme is fluorescent, and fluorescence energy transfer occurs between intrinsic tryptophan and covalently bound bimane in modified enzyme. Both Tyr 115 and Cys 114 are modified, but Tyr 115 is the initial reaction target and its modification correlates with loss of activity toward CDNB. The fact that the activity toward mBBr is retained by the enzyme after modification suggests that rat isozyme 3-3 has two binding sites for mBBr.

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Affinity labeling of glutathione S-transferase, isozyme 4-4, by 4-(fluorosulfonyl)benzoic acid reveals Tyr115 to be an important determinant of xenobiotic substrate specificity

Cited by 43 publications

References 24 publications

Insulin and Glucagon Regulation of GlutathioneS-Transferase Expression in Primary Cultured Rat Hepatocytes

Insulin and Glucagon Regulation of GlutathioneS-Transferase Expression in Primary Cultured Rat Hepatocytes

Probing Subunit Interactions in Alpha Class Rat Liver Glutathione S-Transferase with the Photoaffinity Label Glutathionyl S-[4-(Succinimidyl)benzophenone]

Monobromobimane as an Affinity Label of the Xenobiotic Binding Site of Rat Glutathione S-Transferase 3-3

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