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PubMed1979

Identification of two lithocholic acid-binding proteins. Separation of ligandin from glutathione S-transferase B.

1. Two lithocholic acid-binding proteins in rat liver cytosol, previously shown to have glutathione S-transferase activity, were resolved by CM-Sephadex chromatography. 2. Phenobarbitone administration resulted in induction of both binding proteins. 3. The two proteins had distinct subunit compositions indicating that they are dimers with mol.wts. 44 000 and 47 000. 4. The two lithocholic acid-binding proteins were identified by comparing their elution volumes from CM-Sephadex with those of purified ligandin and glutathione S-transferase B prepared by published procedures. Ligandin and glutathione S-transferase B were eluted separately, as single peaks of enzyme activity, at volumes equivalent to the two lithocholic acid-binding proteins. 5. Peptide 'mapping' revealed structural differences between the two proteins.

Animals↗
PubMed1980

The effect of vitamin A deficiency on hepatic, renal and pulmonary glutathione S-transferase activities in the rat.

Feeding male weanling rats on a vitamin A-deficient diet for 6 weeks resulted in significant increases (44-57%) in glutathione S-aryl-, S-aralkyl- S-alkyl- and S-epoxidetransferase activities in the liver cytosol. Only the S-aralkyl- (27%) and S-alkyltransferase (14%) activities were significantly increased in the kidney as a result of deficiency. There was no effect on any of the pulmonary glutathione S-transferase activities. The increases in hepatic transferase activities were due primarily to increases (25-96%) in the apparent Vmax. There were no changes in the apparant Km of any of the four drug substrates employed. With 3,4-dichloronitrobenzene as the second substrate, the apparent Km for glutathione was increased by over 2-fold in vitamin A-deficient livers as compared with controls. The relationship between these results and enhanced susceptibility to chemical carcinogens in vitamin A deficiency is briefly discussed, and comparison is made between the effects of this nutritional state and pretreatment with drug inducers on the glutathione S-transferases.

Animals↗
PubMed1981

A study of the structures of the YaYa and YaYc glutathione S-transferases from rat liver cytosol. Evidence that the Ya monomer is responsible for lithocholate-binding activity.

The two dimeric lithocholic acid-binding proteins previously identified as ligandin (YaYa) and glutathione S-transferase B (YaYc) were isolated from rat liver cytosol. These proteins have molecular weights of 44000 and 47000 respectively. The recovery of these two proteins from liver was not affected by the addition of the proteinase inhibitor Trasylol. No spontaneous interconversion between these two proteins was observed on storage. YaYa and YaYc proteins yielded peptides of identical molecular weight after limited digestion with Staphylococcus aureus V8 proteinase. Analytical and preparative tryptic-digest peptide 'maps' showed that all the soluble peptides obtained from YaYa protein were also recovered from YaYc protein. Approximately six extra soluble peptides, which were not recovered from YaYa protein, were obtained from the tryptic digest of YaYc protein. Subdigests of the insoluble tryptic-digest 'cores' also resulted in the recovery of identical peptides from both proteins. Evidence is presented that the Ya subunit possessed by both proteins is identical; glutathione S transferase B is a hybrid of ligandin and glutathione S-transferase AA. The Ya monomer is responsible for lithocholate binding.

Animals↗
PubMed1983

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

A purification scheme was devised that resulted in the resolution of a number of basic glutathione S-transferases from rat liver, three of which contained two subunits of molecular mass 23500 Da (i.e. Yb monomers). These were identified as transferases D, C and A by their elution positions from CM-cellulose and their specific activities towards a variety of substrates. Hybridization, immunotitration and peptide 'mapping' experiments demonstrated that transferases D, C and A comprise Yb2Yb2, Yb1Yb2 and Yb1Yb1 subunits.

Amino Acids↗
PubMed1984

Studies on the glutathione S-transferase activity associated with rat liver mitochondria.

The major proportion of rat liver glutathione S-transferase is cytosolic. Carefully washed mitochondria contain 0.25-0.47% of the cytosolic activity. Subfractionation of washed mitochondria using digitonin treatment revealed that glutathione S-transferase release did not parallel that of any of the mitochondrial marker enzymes. Glutathione S-transferase release paralleled that of lactate dehydrogenase, suggesting that these 'mitochondrial' activities are due to loosely bound cytoplasmic forms.

Animals↗
PubMed1985

Different forms of human liver glutathione S-transferases arise from dimeric combinations of at least four immunologically and functionally distinct subunits.

Four immunologically distinct subunits were characterized in glutathione (GSH) S-transferases of human liver. Five cationic enzymes (pI 8.9, 8.5, 8.3, 8.2 and 8.0) have an apparently similar subunit composition, and are dimers of 26 500-Mr (A) and 24 500-Mr (B) subunits. A neutral enzyme, pI 6.8, is a dimer of B-type subunits. One of the anionic enzymes, pI 5.5, is also a dimer of 26 500-Mr subunits. However, the 26 500-Mr subunits of this anionic enzyme form are immunologically distinct from the A subunits of the cationic enzymes, and have been designated as A'. Immunoabsorption studies with the neutral enzyme, BB, and the antibodies raised against the cationic enzymes (AB) indicate that A and B subunits are immunologically distinct. Hybridization in vitro of the A and B subunits of the cationic enzymes (AB) results in the expected binary combinations of AA, AB and BB. Studies with the hybridized enzyme forms indicate that only the A subunits express GSH peroxidase activity. A' subunits have maximum affinity for p-nitrobenzyl chloride and p-nitrophenyl acetate, and the B subunits have highest activity towards 1-chloro-2,4-dinitrobenzene. The other anionic form, pI 4.5, present in liver is a heterodimer of 22 500-Mr (C) and B subunits. The C subunits of this enzyme are probably the same as the 22 500-Mr subunits present in human lung and placental GSH transferases. The distinct immunological nature of B and C subunits was also demonstrated by immunoaffinity and subunit-hybridization studies. The results of two-dimensional polyacrylamide-gel-electrophoretic analyses indicate that in human liver GSH transferases, three charge isomers of Mr 26 500 (A type), two charge isomers of Mr 24 500 (B type) and two charge isomers of Mr 22 500 (C type) subunits are present.

Antibodies↗
PubMed1986

A subclass of glutathione S-transferases as intracellular high-capacity and high-affinity steroid-binding proteins.

The distribution of glucocorticoids incubated with rat liver cytosol preparations or administered in vivo to adrenalectomized rats was analysed by chromatographic procedures. Corticosterone or dexamethasone was co-eluted with Yb-type GSH S-transferases in anion-exchange and gel-permeation chromatography systems, and these glucocorticoids also were bound to Yb forms in analyses by immunoadsorbent and lysyl-GSH affinity matrices. Pretreatment of cytosol with lysyl-GSH to extract GSH S-transferases or incubation with excess bilirubin, which is expected to compete with steroids for binding to the protein, yielded preparations that were devoid of this major steroid-binding component. In mixtures of the multiple rat GSH S-transferases, corticosterone preferentially interacted with Yb forms rather than Ya and Yc subgroups. All of the multiple Yb forms resolved by chromatofocusing procedures retained the steroid-binding capacity. It is suggested that these abundant proteins can account for a considerable share of intracellular glucocorticoid binding and represent a high-affinity non-saturable binding component with potential to function in steroid-hormone metabolism and action.

Animals↗
PubMed1986

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

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.

Amino Acids↗
PubMed1987

Sex-specific constitutive expression of the pre-neoplastic marker glutathione S-transferase, YfYf, in mouse liver.

Hepatic glutathione S-transferase isoenzyme content has been investigated in both sexes of three inbred strains of mice (DBA/2, C3H/He, C57BL6). A polypeptide (Mr 24,800), which is immunologically related to Yf purified from rat lung, was found to be expressed as a major form in all male mouse livers but represented only a minor enzyme form in female mouse liver. Glutathione S-transferases comprising subunits with molecular masses of 25,800 (Ya) or 26,400 (Yb) were present in males and females of the three strains under investigation. Cytosolic isoenzymes from all strains and sexes were purified to apparent homogeneity and no significant inter-strain differences in the properties of the individual forms were observed. In addition, no differences were detected in the microsomal glutathione S-transferase content of the different strains or sexes.

Animals↗
PubMed1991

Inhibition by inorganic anions of glutathione S-transferases from insect and mammalian sources.

Glutathione S-transferases 1-1, 3-3, 3-4 and 4-4 from rat liver and the major glutathione S-transferase from the wax moth (Galleria mellonella) are all inhibited by several simple inorganic anions. For each of 3-3, 3-4 and the insect enzyme, the order of inhibitory potency was ClO4- greater than or equal to SCN- greater than I- greater than NO3- greater than Br-. A more limited range of anions was tested on the isoenzymes 1-1 and 4-4, but the same trend was apparent. Values for Ki ranged from about 200 mM for Cl- to 6 mM for SCN- in the case of the insect enzyme and from 50 mM for Br- to 0.3 mM for SCN- for the rat isoform 3-3. Acetate, F-, SO4(2-) and PO4(3-) were not found to have significant inhibitory properties. The mode of inhibition was characterized as non-competitive in the case of the insect enzyme and rat transferase 1-1, whereas the mode of inhibition was partially non-competitive in the case of the rat isoforms 3-3, 3-4 and 4-4.

Animals↗
PubMed1991

Cysteine-86 is not needed for the enzymic activity of glutathione S-transferase 3-3.

Recombinant glutathione S-transferase 3-3 expressed in Spodoptera frugiperda (SF9) cells with the use of a baculovirus expression system was modified with 1 mM-iodoacetamide. Amino acid analysis indicated that 0.79 +/- 0.15 cysteine residue was modified per enzyme subunit. The S-carbaminomethylated protein retains the GSH-conjugating activity. Glutathione S-transferase 3-3 modified with iodo[14C]acetamide was digested with Achromobacter proteinase I and the resulting peptides were separated by h.p.l.c. The modified peptides were pooled and further digested with Staphylococcus aureus V8 proteinase. Isotope-labelled peptides were isolated and collected for N-terminal sequence analysis. By this procedure, cysteine-86 was identified as the major S-carbaminomethylated residue. Verification of this findings came from the use of site-directed mutagenesis in which this cysteine was replaced by serine (C86S mutant). The C86S mutant is enzymically active. Therefore cysteine-86 is not needed for the conjugation of GSH with electrophilic compounds on glutathione S-transferase 3-3.

Amino Acid Sequence↗
PubMed1991

Immunohistochemical distribution of isoenzymes of glutathione transferase in adult rat adrenal gland before and after hypophysectomy.

The distribution of glutathione transferase subunits 1, 2, 3, 4, 7 and 8 in the different cells of the female and male rat adrenal and the effects of hypophysectomy on these isoenzymes were studied using immunohistochemical methods. All these glutathione transferase subunits, with the exception of subunit 1, were present in the adrenal. Each subunit showed, however, its own characteristic distribution pattern. After hypophysectomy, increased staining for these isoenzymes was generally observed, and this effect was also cell-specific. Staining for subunit 2 increased in intensity in the zona fasciculata and reticularis after hypophysectomy, whereas a decrease was observed in the zona glomerulosa. Staining for subunit 8 was increased in the borderline between the capsule and zona glomerulosa, as well as in medullary chromaffin cells after hypophysectomy. The Mu subunits 3 and 4 increased markedly in fascicular and reticular cells after hypophysectomy and staining for subunit 3 was also increased in the medullary cells. A slight, but more general, increase was observed for subunit 7. We conclude from these experiments that the increases in glutathione transferase subunits observed in the rat adrenal after hypophysectomy are due to increased protein synthesis and/or increased protein stability and not to a selective destruction of cells lacking, or with low levels of, the isoenzymes.

Adrenal Glands↗
PubMed1992

Cloning and heterologous expression of cDNA encoding class alpha rat glutathione transferase 8-8, an enzyme with high catalytic activity towards genotoxic alpha,beta-unsaturated carbonyl compounds.

A cDNA clone, lambda GTRA8, encoding rat glutathione transferase subunit 8 has been isolated from a lambda gt10 rat hepatoma cDNA library. The previously known amino acid sequence of the enzyme was used to design primers for a polymerase chain reaction that yielded a 0.3 kb DNA fragment from the hepatoma library. The 0.3 kb fragment was used as a probe for screening and a 0.9 kb cDNA clone containing a complete open reading frame was obtained. After DNA sequencing and subcloning into an expression vector, the enzyme was expressed in Escherichia coli and purified. Specific activities and kcat./Km values were determined for a number of substrates, including alpha,beta-unsaturated carbonyl compounds. The highest activity was obtained with 4-hydroxyalkenals and with acrolein, genotoxic products of lipid peroxidation. In addition, the rat class Alpha glutathione transferase 8-8 displays high catalytic activity in the reaction between glutathione and the diuretic drug ethacrynic acid, a compound normally considered as a substrate characteristic for class Pi glutathione transferases.

Amino Acid Sequence↗
PubMed1992

Participation of the phenolic hydroxyl group of Tyr-8 in the catalytic mechanism of human glutathione transferase P1-1.

The coding region of cDNA corresponding to human class Pi glutathione transferase P1-1 was amplified by the PCR, subcloned into an expression vector, pKHP1, expressed in Escherichia coli, and characterized. The physicochemical and catalytic properties of recombinant glutathione transferase P1-1 were indistinguishable from those of the enzyme previously isolated from human placenta. The active-site residue Tyr-8 of the wild-type enzyme was converted into Phe by means of oligonucleotide-directed mutagenesis. The mutant enzyme Y8F displayed a 300-fold decrease in specific activity, ascribable mainly to a lowered k(cat.) (or V) value. Kinetic parameters reflecting binding affinity, S0.5 (substrate concn. giving 1/2V) and I50 (concn. of inhibitor giving 50% remaining activity), were only moderately elevated in the mutant enzyme. These results indicate that Tyr-8 contributes primarily to catalysis as such, rather than to binding of the substrates. The dependence of k(cat.)/Km on pH shows an optimum at pH 7.0, defined by acidic and basic ionic dissociation constants with pKa1 = 6.7 and pKa2 = 7.3 respectively. The mutant enzyme Y8F does not display the basic limb of the k(cat.)/Km versus pH profile, but shows a monotonic increase of k(cat.)/Km with an apparent pKa1 of 7.1. The results indicate that the phenolic hydroxyl group of Tyr-8 in un-ionized form, but not the phenolate of Tyr-8, contributes to catalysis by glutathione transferase P1-1.

Base Sequence↗
PubMed1992

Further characterization of hormonal regulation of glutathione transferase in rat liver and adrenal glands. Sex differences and demonstration that growth hormone regulates the hepatic levels.

Immunoblot experiments and reverse-phase h.p.l.c. were used to study the levels of glutathione transferase subunits 1, 2, 3, 4, 6, 7 and 8 in the liver and adrenal of intact and hypophysectomized male and female Sprague-Dawley rats. A sexual dimorphism in the levels of several of these isoenzymes and in their responses to hypophysectomy was demonstrated. In the liver of sham-operated females and males there are differences in glutathione transferase activities and isoenzyme pattern. H.p.l.c. analysis showed higher levels of subunits 1, 3 and 4 in male rats compared with females. In contrast with the pronounced sex differences in sham-operated rats, the isoenzyme patterns of hypophysectomized males and females were very similar. In the adrenal glands, however, a sexual dimorphism became apparent only after hypophysectomy, when the level of subunit 4 was increased 14-fold in the female, whereas the corresponding increase in the male rat was only 2.7-fold. The hepatic pattern of glutathione transferase subunits could be altered by continuous infusion of growth hormone to both sham-operated and hypophysectomized rats of both sexes. This treatment feminized the isoenzyme pattern in sham-operated males and a similar effect was obtained upon treating hypophysectomized rats with thyroxine, cortisone acetate and a continuous infusion of growth hormone.

Adrenal Glands↗
PubMed1995

Characterization of rat spleen prostaglandin H D-isomerase as a sigma-class GSH transferase.

The prostaglandin H D-isomerase of rat immune accessory cells has been purified from spleen by a simple procedure, and its high specificity and activity [Urade, Fujimoto, Ujihara and Hayaishi (1987) J. Biol. Chem. 262, 3820-3825] have been confirmed in an assay coupled to prostaglandin H synthase. The enzyme also decreases the formation of 12[S]-hydroxy-5,8,10-heptadecatrienoic acid formed by the synthase in the presence of GSH and increases the overall rate of arachidonate oxidation. A partial amino acid sequence shows a strong relationship to GSH transferases of parasitic helminths and molluscs, indicating that it is the first example of a vertebrate sigma-class GSH transferase, and suggesting that certain helminth GSH transferases may be involved in prostaglandin synthesis.

Amino Acid Sequence↗
PubMed1996

Arginine-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes.

An Arg-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes (PMNs) was confirmed by the use of diethylamino-(benzylidineamino)guanidine (DEA-BAG) as an ADP-ribose acceptor. Two separate HPLC systems were used to separate ADP-ribosyl-DEA-BAG from reaction mixtures, and its presence was confirmed by electrospray mass spectrometry. ADP-ribosyl-DEA-BAG was produced in the presence of PMNs, but not in their absence. Incubation of DEA-BAG with ADP-ribose (0.1-10 mM) did not yield ADP-ribosyl-DEA-BAG, which indicates that ADP-ribosyl-DEA-BAG formed in the presence of PMNs was not simply a product of a reaction between DEA-BAG and free ADP-ribose, due possibly to the hydrolysis of NAD+ by an NAD+ glycohydrolase. The assay of mono(ADP-ribosyl)transferase with agmatine as a substrate was modified for intact PMNs, and the activity was found to be approx. 50-fold lower than that in rabbit cardiac membranes. The Km of the enzyme for NAD+ was 100.1 30.4 microM and the Vmax 1.4 0.2 pmol of ADP-ribosylagmatine/h per 10(6) cells. The enzyme is likely to be linked to the cell surface via a glycosylphosphatidylinositol anchor, since incubation of intact PMNs with phosphoinositol-specific phospholipase C (PI-PLC) led to a 98% decrease in mono(ADP-ribosyl)transferase activity in the cells. Cell surface proteins were labelled after exposure of intact PMNs to [32P]NAD+. Their molecular masses were 79, 67, 46, 36 and 26 kDa. The time course for labelling was non-linear under these conditions over a period of 4 h. The labelled products were identified as mono(ADP-ribosyl)ated proteins by hydrolysis with snake venom phosphodiesterase to yield 5'-AMP.

ADP Ribose Transferases↗
PubMed1996

The distribution of theta-class glutathione S-transferases in the liver and lung of mouse, rat and human.

Two murine Theta-class glutathione S-transferases (GSTs), mGSTT1 and mGSTT2, have been cloned and sequenced. The murine cDNAs, together with the published sequences of the rat and human enzymes, were used to design oligonucleotide probes in order to determine the distribution of mRNA for these enzymes in the liver and lung of rat, mouse and human. The mRNA distribution was compared with that of enzyme protein determined with an antibody to rat GSTT2-2. Both the antibody and the oligonucleotide probes gave the same distribution patterns. Both enzymes were present at significantly higher concentrations in mouse tissues than in rat or human tissues. In mouse liver, both enzymes were localized in specific cell types and in nuclei. Although the distribution of GSTT2-2 in rat liver was similar to that seen in the mouse, GSTT1-1 was not localized in a specific cell type or in the nuclei of either rat or human liver. In the lungs, very high concentrations of the Theta enzymes were present in mouse-lung Clara cells and ciliated cells, with much lower levels in the Clara cells only of rat lung. Low levels of human transferase GSTT1-1 were detected in a small number of Clara cells and ciliated cells at the alveolar/ bronchiolar junction. The relative activities between species, and the cellular and sub-cellular distribution within the liver and lungs of each species, provides an explanation for the species-specificity of methylene chloride, a mouse-specific carcinogen activated by glutathione S-transferase GSTT1-1.

Animals↗