Rat liver glutathione <i>S</i>-transferases. A study of the structure of the basic YbYb-containing enzymes

Hayes, John D.

doi:10.1042/bj2130625

Cited by 50 publications

(44 citation statements)

References 24 publications

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“…The kinetic data in Table 2 suggest a greater specificity for GSH. This is not surprising in view of the high intracellular concentration of GSH estimated to be as high as 10 mM [36], which will keep GSH binding site occupied.…”

Section: Discussionmentioning

confidence: 89%

Purification and catalytic properties of glutathione transferase from the hepatopancreas of crayfishmacrobrachium vollenhovenii (herklots)

Adewale¹,

Afolayan²

2005

J. Biochem. Mol. Toxicol.

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Glutathione transferase from the hepatopancreas of fresh water crayfish Macrobrachium vollenhovenii was purified to apparent homogeneity by ion-exchange chromatography on DEAE-cellulose and by gel filtration on Sephadex G-100. The enzyme appeared to be a homodimer with molecular weight (Mr) of 46.0 +/- 1.4 kDa and a subunit Mr of 24.1 +/- 0.35 kDa. Chromatofocusing of the apparently pure enzyme revealed microheterogeneity and resolved it into two isozymic peaks, which were eluted at pH 8.36 and 8.22 respectively. Inhibition studies showed that the I50 value for cibacron blue, S-hexylglutathione, hematin, and N-ethylmaleimide (NEM) were 0.01 microM, 340 microM, 5 microM and 33 mM respectively. Out of the several substrates tested, only 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole could be conjugated with glutathione. Chemical modification studies with DTNB revealed that two sulphydryl groups per dimer were essential to the activity of the enzymes. On the basis of structural and catalytic characteristics, M. vollenhovenii GST seems close, tentatively, to the omega and zeta classes of GST. Initial-velocity studies of the enzyme are consistent with a steady-state random kinetic mechanism. Denaturation and renaturation studies with guanidine HCl (Gdn-HCl) revealed that though low Gdn-HCl concentrations (less than 0.5 M) denatured the enzyme, the enzyme was able to renature completely (100%). At higher concentration of the denaturant (0.5-4 M), refolding studies indicated that complete renaturation was not achieved. The extent of renaturation was however a function of protein concentration. Our results are consistent with a three-state unfolding process.

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Section: Discussionmentioning

confidence: 89%

Purification and catalytic properties of glutathione transferase from the hepatopancreas of crayfishmacrobrachium vollenhovenii (herklots)

Adewale¹,

Afolayan²

2005

J. Biochem. Mol. Toxicol.

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“…Sci. USA 82 (1985) several investigators (30)(31)(32). It is possible that GSHTase-P is identical with the tissue-specific GSHTase of Mr 24,000, which was found by Tu and co-workers (29,30).…”

Section: Discussionmentioning

confidence: 99%

Purification, induction, and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical hepatocarcinogenesis.

Satoh¹,

Kitahara²,

Soma³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

472

246

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A polypeptide of Mr 26,000 and pI 6.7 that was markedly increased in rat livers bearing hyperplastic nodules (HNs) induced by chemical carcinogens was identified immunochemically as the subunit of neutral glutathione (GSH) transferase (GSHTase; RX:glutathione R-transferase, EC 2.5.1.18; also called GSH S-transferase) purified from placenta (GSHTase-P) and was demonstrated immunohistochemically to be localized in preneoplastic foci and HNs. In the present study, GSHTase-P has been purified from the HN-bearing liver, and the distribution and inducibility have been examined quantitatively using anti-GSHTase-P antibody. Elevation of GSHTase-P in the HN-bearing livers was also confirmed by in vitro translation of mRNAs isolated from the HN-bearing livers. The purified GSHTase-P was homogeneous in size but had two charge isomers on two-dimensional gel electrophoresis. In normal tissues, including liver, placenta, and fetal liver, the protein content of GSHTase-P was generally low but was significantly high in kidney and pancreas. In contrast, the amount of GSHTase-P in HN-bearing livers (primary hepatomas) and transplantable Morris hepatoma 5123D were several 10-fold higher than that in normal liver but were undetectably low in transplantable Yoshida ascites hepatoma AH 130. Different from ordinary drug-metabolizing enzymes, GSHTase-P was uninducible by administration of drugs and carcinogens prior to appearance of the preneoplastic foci and HNs. In addition, species specificity of GSHTase-P was low as it was crossreactive among rat, hamster, and human.Much attention has been focused on the morphological, histochemical, and biochemical properties of the preneoplastic cells such as enzyme-altered foci and hyperplastic nodules (HNs) induced at early stages of chemical hepatocarcinogenesis (1-5). Among (pre)neoplastic markers so far found, epoxide hydrase (6) tocarcinogenesis. In our studies of isozymic alterations during chemical hepatocarcinogenesis, especially of drugmetabolizing enzymes (7, 11-15), we observed by twodimensional gel electrophoretic analysis of the cytosolic proteins of the HN-bearing rat livers that a polypeptide of Mr 26,000 and pI 6.7 [designated polypeptide (26/6.7)] increased most markedly together with three other proteins. This polypeptide was identified immunochemically as the subunit of a neutral GSHTase that was purified from rat placenta. This placental form (GSHTase-P) was demonstrated to be localized in HNs and in foci positive for y-glutamyltransferase (GluTase; y-glutamyl transpeptidase, EC 2.3.2.2) (14).In the present study, GSHTase-P has been purified from HN-bearing rat livers, some chemical properties have been characterized, and the content of this protein in normal, preneoplastic, and neoplastic tissues and its inducibility in rat livers by drugs and carcinogens have been examined.MATERIALS AND METHODS Animals. Male Sprague-Dawley rats, weighing 150-160 g, were obtained from Charles River-Oriental Yeast (Tokyo).Chemicals. Chemicals were obtained as follows; diethylnitrosami...

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“…It is related, but not identical, to transferases 3-3 and 3-4. In this context, transferase X is similar (possibly identical) to the isozyme called glutathione transferase 4-4 [7,81 and to transferase Id [6]. Glutathione transferase 5-5 (or E), another type of hepatic near-neutral transferases, has an isoelectric point of 7.0 [24].…”

Section: Nature Of Neur-neutral Form Of Cardiac Glutathione Trans Fermentioning

confidence: 99%

The major isozyme of rat cardiac glutathione transferases. Its correspondence to hepatic transferase X

et al. 1986

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1. A major isozyme of rat heart glutathione transferase was purified to homogeneity by Sephadex G-200 gel filtration, ammonium sulfate precipitation, CM-cellulose chromatography and affinity chromatography on Shexylglutathione-linked Sepharose 6 B.2. The purified isozyme was a dimer with an apparent relative molecular mass of 50000 composed of two Ybsize subunits (Mr = 26500). The isozyrne is immunologically related to rat liver glutathione transferase X and 3-3, especially closely to transferase X, and no immunological cross-reactivity with subunits 1 and 2 of hepatic glutathione transferases was observed. The isoelectric point (PI = 6.9) of the isozyme was identical with and the substrate specificity was very similar to transferase X. Thus, the cardiac near-neutral isozyme is considered to be identical to glutathione transferase X recognized in rat liver.3. The amount of this near-neutral isozyme estimated to be present in heart tissue is 70 pg/g. The isozyme has relatively high activities towards a$-unsaturated carbonyl compounds such as trans-4-phenyl-3-buten-2-one and trmns-4-hydroxynon-2-enal. The latter is a cytotoxic product resulting from lipid peroxidation of polyunsaturated fatty acids, and the cardiac isozyme may play a physiologically significant role with glutathione conjugation of this compound.4. In addition to the near-neutral isozyme, acidic forms with isoelectric points of 4.9, 5.2 and 5.5 were partially purified; some of them are considered to consist of subunits immunologically related to transferase X.

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Rat liver glutathione S-transferases. A study of the structure of the basic YbYb-containing enzymes

Cited by 50 publications

References 24 publications

Purification and catalytic properties of glutathione transferase from the hepatopancreas of crayfishmacrobrachium vollenhovenii (herklots)

Purification and catalytic properties of glutathione transferase from the hepatopancreas of crayfishmacrobrachium vollenhovenii (herklots)

Purification, induction, and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical hepatocarcinogenesis.

The major isozyme of rat cardiac glutathione transferases. Its correspondence to hepatic transferase X

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