Rat lung glutathione S-transferases: Subunit structure and the interrelationship with the liver enzymes

Partridge, Catherine A.; Singh, S.V.; Hong, Thomas D.; Theodore, C; Dao, Dat D.; Awasthi, Yogesh C.

doi:10.1016/0020-711x(85)90208-3

Cited by 16 publications

(6 citation statements)

References 31 publications

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“…Prior to analysis, the fusion protein was cleaved with thrombin to remove the GST moiety, which is capable of homodimerization (16). Full-length recombinant Eps15 was then incubated with the water-soluble heterobifunctional cross-linker BS 3 .…”

Section: Resultsmentioning

confidence: 99%

Eps15 Is Constitutively Oligomerized Due to Homophilic Interaction of Its Coiled-coil Region

Tebar¹,

Confalonieri²,

Carter³

et al. 1997

Journal of Biological Chemistry

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Eps15 is a member of an emerging family of proteins containing a novel protein/protein interaction domain, the EH domain, of as yet unknown function. Recent findings of Eps15 association with clathrin adaptor complex AP-2 and its localization in clathrin-coated pits have implicated Eps15 in the regulation of vesicle trafficking. Here we show that Eps15 exists in several multimeric states in vivo. When purified recombinant Eps15 or lysates of NIH 3T3 cells were treated with cross-linking reagents, covalent dimers of Eps15 and larger covalent multimers were detected in high yield. Large Eps15 oligomers co-immunoprecipitated with AP-2 at an efficiency higher than that of Eps15 dimers. Furthermore, cross-linking of the membrane-bound fraction of Eps15 in mildly permeabilized cells was as efficient as that of the cytosolic fraction. Size-exclusion column chromatography of recombinantly produced Eps15 and of total cell lysates was performed to examine the equilibrium ratio of the monomers versus the aggregated forms of Eps15. These experiments showed that essentially all the Eps15 was aggregated, whereas monomers of Eps15 could be obtained only under strong denaturing conditions. To map the region of Eps15 responsible for dimerization, fusion proteins corresponding to the three structural domains of Eps15 were prepared. Cross-linking analysis revealed that the central portion of Eps15, which possesses a coiled-coil region (residues 321-520), serves as the interacting interface. The possibility that hetero-oligomeric complexes of Eps15 dimers and AP-2 function during the recruitment of proteins into coated pits is discussed.The Eps15 protein was originally discovered as a phosphorylation substrate of the epidermal growth factor receptor kinase (1). Subsequent studies revealed that Eps15 is constitutively associated with the plasma membrane clathrin adaptor protein complex AP-2 (2). Immunomorphological analysis demonstrated that membrane-bound Eps15 is mainly associated with the plasma membrane clathrin-coated pits and vesicles (3). Moreover, Eps15 is distributed asymmetrically within the coat, mostly at the rim of the pits and at the neck of the budding vesicles. All these findings suggested that Eps15 might play a role in clathrin-dependent endocytosis.Further evidence suggesting a role for Eps15 in protein trafficking comes from the analysis of the primary structure of Eps15 and its homology to other proteins. The predicted amino acid sequence of Eps15 identifies at least three structural domains (1, 4). The amino-terminal region is composed of three relatively conserved repeats of ϳ70 amino acids referred to as EH domains (for Eps15 homology). The central domain of Eps15 presents the characteristic heptad repeats of coiled-coil proteins. The carboxyl-terminal domain displays several AspPro-Phe (DPF) 1 repeats, proline-rich sequences capable of interacting with SH3 domains (5), and the binding site for the ␣-subunit of AP-2 (6, 7). Importantly, an EH domain is found in End3p, a protein required for endocytosis of ␣-f...

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Section: Resultsmentioning

confidence: 99%

Eps15 Is Constitutively Oligomerized Due to Homophilic Interaction of Its Coiled-coil Region

Tebar¹,

Confalonieri²,

Carter³

et al. 1997

Journal of Biological Chemistry

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“…Even though GST activity towards the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB) is increased in mouse lung on the administration of BHA (Benson et al, 1979), it is not known whether the activity of this enzyme system towards the PAH epoxides is also affected. This is pertinent because the multiple forms of GST characterized from rat (Partridge et al, 1985;Singh et al, 1984) or human lung (Partridge et al, 1984) differ significantly among each other in their binding properties and substrate specificities, and in rat tissues these isoenzymes are differentially induced by BHT (Awasthi et al, 1984;Singh et al, 1985a). It should also be pointed out that, owing to the inter-species differences in the composition and properties of GST isoenzymes in mammalian lung (Partridge et al, 1984(Partridge et al, , 1985Singh et al, 1984), the characteristics of rat lung GST cannot be extrapolated to the mouse model that has been used to study the protective role of antioxidants against chemical carcinogenesis.…”

Section: Introductionmentioning

confidence: 99%

“…This is pertinent because the multiple forms of GST characterized from rat (Partridge et al, 1985;Singh et al, 1984) or human lung (Partridge et al, 1984) differ significantly among each other in their binding properties and substrate specificities, and in rat tissues these isoenzymes are differentially induced by BHT (Awasthi et al, 1984;Singh et al, 1985a). It should also be pointed out that, owing to the inter-species differences in the composition and properties of GST isoenzymes in mammalian lung (Partridge et al, 1984(Partridge et al, , 1985Singh et al, 1984), the characteristics of rat lung GST cannot be extrapolated to the mouse model that has been used to study the protective role of antioxidants against chemical carcinogenesis. Therefore, to address the question whether or not GSTs are involved in the anti-neoplastic activity of these antioxidants, it is essential that the isoenzyme/ subunit composition of mouse lung GST be determined, the activities of the isoenzymes towards PAH metabolites be estimated, and the effect of the antioxidant on each of the isoenzymes be investigated.…”

Section: Introductionmentioning

confidence: 99%

Glutathione S-transferases of mouse lung. Selective binding of benzo[a]pyrene metabolites by the subunits which are preferentially induced by t-butylated hydroxyanisole

Singh¹,

Creadon²,

Das³

et al. 1987

Biochemical Journal

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Six isoenzymes of glutathione S-transferase (GST) present in mouse lung have been purified and characterized. GST I (pI 9.8) is a dimer of Mr-26,500 subunits and GST II is a heterodimer of Mr-26,500 and -22,000 subunits, and GST III (pI 7.9) and IV (pI 6.4) are dimers of Mr-24,500 subunits. GST V (pI 5.7) is a heterodimer of Mr-24,500 and -23,000 subunits, whereas GST VI (pI 4.9) is a dimer of Mr-23,000 subunits. Immunological studies indicate that the Mr-24,500 subunits present in GST III (pI 7.9) are distinct from those present in GST IV (pI 6.4) and V (pI 5.7). Structural and immunological studies provide evidence that at least five distinct types of subunits in their different binary combinations give rise to various GST isoenzymes of mouse lung. These isoenzymes express varying degrees of catalytic activities towards a wide range of electrophilic substrates including benzo[a]pyrene 7,8-oxide and benzo[a]pyrene 4,5-oxide. The dietary antioxidant t-butylated hydroxyanisole (BHA) preferentially induces GST II and III. Also, these two isoenzymes selectively bind benzo[a]pyrene (B[a]P) metabolites, indicating that they play an important physiological role in the detoxification of B[a]P metabolites. The preferential induction of the GST isoenzymes involved in the detoxification of activated B[a]P metabolites indicates that the anti-neoplastic activity of BHA against B[a]P-induced neoplasia in mouse lung [Wattenberg (1973) J. Natl. Cancer Inst. 50, 1541-1544] may be due to the enhanced detoxification of B[a]P metabolites.

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“…More recently, an increasing number of papers have reported the isolation and characterization of anionic isoenzymes (i.e. those with acidic pl), which bind to the anionic exchanger DEAE-cellulose (Awasthi et al, 1980;Friedberg et al, 1983;Guthenberg et al, 1979Guthenberg et al, , 1983Marcus et al, 1978;Maruyama et al, 1983;Partridge et al, 1984Partridge et al, , 1985Reddy et al, 1983;Saneto et al, 1980Saneto et al, , 1982Warholm et al, 1983). The major anionic glutathione S-transferase of bovine ciliary body was purified and characterized in the present work.…”

Section: Discussionmentioning

confidence: 99%

Purification and properties of anionic glutathione S-transferase from bovine ciliary body

Shichi¹,

O'Meara²

1986

Biochemical Journal

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An anionic glutathione S-transferase was purified from bovine ciliary body by DEAE-agarose chromatography and affinity chromatography on GSH-agarose and Orange A. The enzyme accounts for about 25% of total soluble glutathione S-transferase activity of the tissue. The purified enzyme has a molecular mass of about 50,000 Da and is composed of two identical subunits of about 25,000 Da. The enzyme has a pI of 5.8. The enzyme conjugates GSH with 1-chloro-2,4-dinitrobenzene, p-nitrobenzyl chloride, 1,2-epoxy-3-(p-nitrophenyl)propane, 1,2-dinitrobenzene and 3,4-dinitrobenzoic acid. The Km values for 1-chloro-2,4-dinitrobenzene and GSH are 0.40 mM and 0.57 mM respectively. Haematin is a non-competitive inhibitor (Ki = 4.5 microM) when tested with various concentrations of 1-chloro-2,4-dinitrobenzene. The enzyme shows no glutathione peroxidase activity with either H2O2 or cumene peroxide as substrate. On the basis of substrate specificities, pI values, amino acid composition and peptide maps, it is concluded that the ciliary-body enzyme is probably identical with the anionic form of glutathione S-transferase from bovine lens and liver.

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Rat lung glutathione S-transferases: Subunit structure and the interrelationship with the liver enzymes

Cited by 16 publications

References 31 publications

Eps15 Is Constitutively Oligomerized Due to Homophilic Interaction of Its Coiled-coil Region

Eps15 Is Constitutively Oligomerized Due to Homophilic Interaction of Its Coiled-coil Region

Glutathione S-transferases of mouse lung. Selective binding of benzo[a]pyrene metabolites by the subunits which are preferentially induced by t-butylated hydroxyanisole

Purification and properties of anionic glutathione S-transferase from bovine ciliary body

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