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At least 721 records · Page 40Linked to original sources
PubMed1983

Glutathione S-transferase activities in the yellow-fever mosquito [Aedes aegypti (Louisville)] during growth and aging.

Our previous findings [Hazelton & Lang (1978) Fed. Proc. Fed. Am. Soc. Exp. Biol. 37(6), 2378 (abstr.)] demonstrated aging-specific changes in glutathione concentrations in the yellow-fever mosquito [Aedes aegypti (Louisville)]. A possible mechanism could be increased utilization via glutathione S-transferase. Thus glutathione S-transferase activities were measured in mosquito samples from the entire life span, including growth, maturity and senescence. Methods were validated for the quantitative determination of transferase activities with 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-3,4-dinitrobenzene (CDNB) as second substrates. Marked changes occurred during the life span, and the profiles for both DCNB and CDNB activities were identical. The activities increased throughout larval development and reached a maximum in the metamorphosis stage. The activities decreased at the end of metamorphosis in the 5-day-old adult, reached a plateau during maturity (5-20 days), and then decreased 31% (P less than 0.007) during senescence (after 33 days). This senescence-specific decrease occurred in both sexes and was localized in the abdominal region. Further kinetic analyses indicated that the lower enzyme activities were most likely due to lower amounts of active enzyme rather than a change in kinetic properties. These findings indicate that the capacity for GSH utilization via glutathione S-transferase is diminished with aging. This does not explain our previously observed decreases in GSH, but the results suggest that GSH-linked detoxification would be impaired during senescence.

Aedes↗
PubMed1984

Purification and properties of glutathione S-transferases from larvae of Wiseana cervinata.

The glutathione S-transferases from the porina moth, Wiseanna cervinata, were purified by affinity chromatography, cation-exchange chromatography and preparative isoelectrofocusing. The major transferase (IV) was purified to homogeneity by a factor of 530-fold with a yield of 83%. Other transferases present were purified to a smaller degree (approx. 50-fold) to a stage of near-homogeneity. The transferases examined all had Mr values about 45 000-50 000. They appeared to be homodimers of either of two types of subunit, of Mr 22 800 and 24 600. Enzymes consisting of the different types of subunit were not immunologically cross-reactive. The major enzyme fractions separated by cation-exchange chromatography were both active with 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, ethacrynic acid and iodomethane, but were inactive with 4-nitropyridine N-oxide, 1,2-epoxy-3-(p-nitrophenoxy)propane, bromosulphophthalein and p-nitrobenzyl chloride. The kinetics of the enzyme-catalysed reaction with enzyme IV were non-Michaelean with respect to both substrates. Both products were inhibitory. The results appear to be compatible with a random steady-state mechanism. It is concluded that these enzymes are very similar, in their physical and chemical constitution, in their catalytic properties and in their relationships with each other, to those enzymes that have been isolated from vertebrate organisms.

Amino Acids↗
PubMed1984

Selective induction of glutathione S-transferase D in rat testis by phenobarbital.

Testis cytosol is shown to contain the Yb2Yb2 -homodimer glutathione S-transferase D in addition to the previously described glutathione S-transferases A ( Yb1Yb1 ) and C ( Yb1Yb2 ). Treatment of rats with phenobarbital induces the level of glutathione S-transferase D in testis with no increase in the activities of glutathione S-transferases A and C. This result indicates a specific induction of the Yb2 subunit in testis, in contrast with the situation in rat liver, where phenobarbital specifically induces the Yb1 subunit.

Animals↗
PubMed1984

Human glutathione S-transferases. Characterization of the anionic forms from lung and placenta.

Anionic glutathione S-transferases were purified from human lung and placenta. Chemical and immunochemical characterization, including polyacrylamide-gel electrophoresis, gave strong evidence that the anionic lung and placental enzymes are chemically similar, if not identical, proteins. The electrophoretic mobilities of both proteins were identical in conventional alkaline gels as well as in gels containing sodium dodecyl sulphate. Gel filtration of the intact active enzyme established an Mr value of 45000; however, with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under dissociating conditions a subunit Mr of 22500 was obtained. Amino acid sequence analysis of the N-terminal region of the placental enzyme revealed a single polypeptide sequence identical with that of lung. Results obtained from immunoelectrophoresis, immunotitration, double immunodiffusion and rocket immunoelectrophoresis also indicated the anionic lung and placental enzymes to be closely similar. The chemical similarity of these two proteins was further supported by protein compositional analysis and fragment analysis after chemical hydrolysis. Immunochemical comparison of the anionic lung and placental enzymes with human liver glutathione S-transferases revealed cross-reactivity with the anionic omega enzyme, but no cross-reactivity was detectable with the cationic enzymes. Comparison of the N-terminal region of the human anionic enzyme with reported sequences of rat liver glutathione S-transferases gave strong evidence of chemical similarity, indicating that these enzymes are evolutionarily related. However, computer analysis of the 30-residue N-terminal sequence did not show any significant chemical similarity to any other reported protein sequence, pointing to the fact that the glutathione S-transferases represent a unique class of proteins.

Amino Acid Sequence↗
PubMed1985

Glutathione S-transferases of human brain. Evidence for two immunologically distinct types of 26500-Mr subunits.

Human brain contains one cationic (pI8.3) and two anionic (pI5.5 and 4.6) forms of glutathione S-transferase. The cationic form (pI8.3) and the less-anionic form (pI5.5) do not correspond to any of the glutathione S-transferases previously characterized in human tissues. Both of these forms are dimers of 26500-Mr subunits; however, immunological and catalytic properties indicate that these two enzyme forms are different from each other. The cationic form (pI8.3) cross-reacts with antibodies raised against cationic glutathione S-transferases of human liver, whereas the anionic form (pI5.5) does not. Additionally, only the cationic form expresses glutathione peroxidase activity. The other anionic form (pI4.6) is a dimer of 24500-Mr and 22500-Mr subunits. Two-dimensional gel electrophoresis demonstrates that there are three types of 26500-Mr subunits, two types of 24500-Mr subunits and two types of 22500-Mr subunits present in the glutathione S-transferases of human brain.

Amino Acids↗
PubMed1985

Interactions with glutathione S-transferases of porphyrins used in photodynamic therapy and naturally occurring porphyrins.

Several naturally occurring porphyrins and porphyrins used in photodynamic therapy inhibit glutathione S-transferase isoenzymes either purified from rat liver or lung or in cytosol from normal and from cancerous (Morris 7288C hepatoma) liver. Although differences occur in the type and amount of transferases in normal and cancerous liver and in the liver of rats bearing an extrahepatic tumour, these enzymes are potential binding sites for porphyrins. Porphyrin structure is an important factor in determining the affinity of binding, as shown by the relative inhibitory effectiveness. Of the dicarboxylic porphyrins in the mixture used clinically, OO'-diacetylhaematoporphyrin and monohydroxyethylmonovinyldeuteroporphyrin are more effective inhibitors than haematoporphyrin and protoporphyrin IX. Of the naturally occurring porphyrins the order of effectiveness is protoporphyrin IX (dicarboxylic) greater than coproporphyrin (tetracarboxylic) greater than uroporphyrin (octacarboxylic) and type I greater than type III isomers of both uroporphyrin and coproporphyrin, and the synthetic tetra-meso-phenylporphinetetrasulphonate is a better inhibitor (apparent Ki = 250 nM) than coproporphyrin, which contains a comparable number of negative charges. In addition, iron-porphyrin chelates are more effective inhibitors of the transferases, with 25-fold decrease in Ki value, than the free porphyrins. These results indicate that one means whereby porphyrins accumulate in tissues is the occupation of intracellular binding sites, such as the transferases. Since porphyrins inhibit the activity of these important detoxifying enzymes, there will be metabolic consequences to the cell.

Animals↗
PubMed1985

Acidic glutathione S-transferases of rat testis.

In most organs of the rat the predominant forms of glutathione S-transferase have alkaline (greater than 7.0) pI values. In contrast, in the cytosol from rat testes almost 50% of the transferase activity is due to isoenzymes with acidic (less than 7.0) pI values. We have purified three acidic forms of glutathione S-transferase from rat testis cytosol. One form accounted for more than 90% of the enzymic activity in the acidic fraction. This major form was a homodimer of a new subunit, termed Yt. This subunit had an electrophoretic mobility that was different from the subunits that form the alkaline transferases. In addition, functional and immunological studies were consistent with the unique nature of the Yt subunit. The two minor acidic enzymes of rat testis appeared to be heterodimers of the Yt subunit and a subunit with an electrophoretic mobility identical with that of the Yb subunit present in some alkaline enzymes.

Amino Acids↗
PubMed1986

Differential tissue expression of the glutathione transferase multigene family.

The content of GSH transferase mRNAs in poly(A)-containing RNA isolated from eight rat tissues was examined by immunoprecipitation of cell-free translation products and by Northern blotting. Considerable tissue-specific distribution and heterogeneity of immunoprecipitable GSH transferase subunits 1 and 2 synthesized in vitro was observed. These results were confirmed by Northern blotting using a 32P-labelled subunit 1 cDNA probe. The same probe, used in a Southern blot analysis of genomic DNA, provided confirmation that GSH transferase subunits 1 and 2 comprise a multigenic family in the rat. The results show that the selection of cDNA clones coding for chosen subunits can be made easier by making use of qualitative and quantitative tissue differences in GSH transferase mRNAs.

Animals↗
PubMed1988

Stereoselectivity of rat liver glutathione transferase isoenzymes for alpha-bromoisovaleric acid and alpha-bromoisovalerylurea enantiomers.

The stereoselectivity of purified rat GSH transferases towards alpha-bromoisovaleric acid (BI) and its amide derivative alpha-bromoisovalerylurea (BIU) was investigated. GSH transferase 2-2 was the only enzyme to catalyse the conjugation of BI and was selective for the (S)-enantiomer. The conjugation of (R)- and (S)-BIU was catalysed by the isoenzymes 2-2, 3-3 and 4-4. Transferase 1-1 was less active, and no catalytic activity was observed with transferase 7-7. Isoenzymes 1-1 and 2-2 of the Alpha multigene family preferentially catalysed the conjugation of the (S)-enantiomer of BIU (and BI), whereas isoenzymes 3-3 and 4-4 of the Mu multigene family preferred (R)-BIU. The opposite stereoselectivity of conjugation of BI and BIU previously observed in isolated rat hepatocytes and the summation of activities of enzymes known to be present in hepatocytes on the basis of present data are in accord.

Animals↗
PubMed1990

Chemical modification of rat liver microsomal glutathione transferase defines residues of importance for catalytic function.

Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5'-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.

Animals↗
PubMed1995

Glutathione analogues as novel inhibitors of rat and human glutathione S-transferase isoenzymes, as well as of glutathione conjugation in isolated rat hepatocytes and in the rat in vivo.

Inhibitors of rat and human Alpha- and Mu-class glutathione S-transferases that effectively inhibit the glutathione (GSH) conjugation of bromosulphophthalein in the rat liver cytosolic fraction, isolated rat hepatocytes and in the rat liver in vivo have been developed. The GSH analogue (R)-5-carboxy-2-gamma-(S)-glutamylamino-N-hexylpentamide [Adang, Brussee, van der Gen and Mulder (1991) J. Biol. Chem. 266, 830-836] was used as the lead compound. To obtain more potent inhibitors, it was modified by replacement of the N-hexyl moiety by N-2-heptyl and by esterification of the 5-carboxy group with ethyl and dodecyl groups. In isolated hepatocytes, the branched N-2-heptyl derivatives were stronger inhibitors of GSH conjugation of bromosulphophthalein than the N-hexyl derivatives. The ethyl ester compounds were more efficient than the corresponding unesterified derivatives. The dodecyl ester of the N-2-heptyl analogue was the most effective inhibitor in isolated hepatocytes, but was relatively toxic in vivo. However, the corresponding ethyl ester was a potent in vivo inhibitor: GSH conjugation of bromosulphophthalein (as assessed by biliary excretion of the conjugate) was decreased by 70% after administration of a dose of 200 mumol/kg. The isoenzyme specificity of the inhibitors towards purified rat and human glutathione S-transferases was also examined. The unesterified compounds were more potent than the esterified analogues, and inhibited Alpha- and Mu-class isoenzymes of both rat and human glutathione S-transferase (Ki range 1-40 microM). Other GSH-dependent enzymes, i.e. GSH peroxidase, GSH reductase and gamma-glutamyltranspeptide, were not inhibited. Thus (R)-5-ethyloxycarbonyl-2-gamma-(S)-glutamylamino-N-2-hept ylpentamide, the in vivo inhibitor of GSH conjugation, may be useful in helping to assess the role of the Alpha and Mu classes of glutathione S-transferases in cellular biochemistry, physiology and pathology.

Animals↗
PubMed1995

Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli.

The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.

Animals↗
PubMed1995

Amino acid sequencing, molecular cloning and modelling of the chick liver class-theta glutathione S-transferase CL1.

Glutathione S-transferase CL1-2 heterodimers purified from 1-day-old chick livers were digested with Achromobacter proteinase I. The resulting fragments were separated for amino acid sequence analysis. Oligonucleotide probes were constructed based on sequence similarity to class-Theta glutathione S-transferases for PCR using a chicken liver cDNA library as template. A full-length clone (1725 bp) encoding a polypeptide comprising 261 amino acids was isolated. Including conservative substitutions, this protein has 70-73% sequence similarity with other mammalian class-Theta glutathione S-transferases. Based on known X-ray crystal structures of class-Alpha, -Mu and -Pi glutathione S-transferases, a model is constructed for the N-terminal 232 residues of CL1.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of prostaglandin-H E-isomerase, a sigma-class glutathione S-transferase, from Ascaridia galli.

Comparison of partial primary sequences of sigma-class glutathione S-transferases (GSH) of parasitic helminths and a GSH-dependent prostaglandin (PG)-H D-isomerase of rat immune accessory cells suggested that some of the helminth enzymes may also be involved in PG biosynthesis [Meyer and Thomas (1995) Biochem. J. 311, 739-742]. A soluble GSH transferase of the parasitic nematode Ascaridia galli has now been purified which shows high activity and specificity in the GSH-dependent isomerization of PGH to PGE, comparable to that of the rat spleen enzyme in its isomerization of PGH to PGD, and similarly stimulates the activity of prostaglandin H synthase. The enzyme subunit is structurally related to the rat spleen enzyme and sigma-class GSH transferases of helminths according to the partial primary sequence. The data support the hypothesis that some sigma-class GSH transferases of helminth parasites are involved in PG biosynthesis which, in the case of PGE, is likely to be associated with the subversion or suppression of host immunity. A PG-H E-isomerase of comparable specificity and activity has not previously been isolated.

Amino Acid Sequence↗
PubMed2002

Propionate CoA-transferase from Clostridium propionicum. Cloning of gene and identification of glutamate 324 at the active site.

Propionate CoA-transferase from Clostridium propionicum has been purified and the gene encoding the enzyme has been cloned and sequenced. The enzyme was rapidly and irreversibly inactivated by sodium borohydride or hydroxylamine in the presence of propionyl-CoA. The reduction of the thiol ester between a catalytic site glutamate and CoA with borohydride and the cleavage by hydroxylamine were used to introduce a site-specific label, which was followed by MALDI-TOF-MS. This allowed the identification of glutamate 324 at the active site. Propionate CoA-transferase and similar proteins deduced from the genomes of Escherichia coli, Staphylococcus aureus, Bacillus halodurans and Aeropyrum pernix are proposed to form a novel subclass of CoA-transferases. Secondary structure element predictions were generated and compared to known crystal structures in the databases. A high degree of structural similarity was observed between the arrangement of secondary structure elements in these proteins and glutaconate CoA-transferase from Acidaminococcus fermentans.

Amino Acid Sequence↗
PubMed2001

The pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the peptidyl transferase centre on the ribosome.

The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs are strong inhibitors of peptidyl transferase and interact with domain V of 23S RNA, giving clear chemical footprints at nucleotides A2058-9, U2506 and U2584-5. Most of these nucleotides are highly conserved phylogenetically and functionally important, and all of them are at or near the peptidyl transferase centre and have been associated with binding of several antibiotics. Competitive footprinting shows that tiamulin and valnemulin can bind concurrently with the macrolide erythromycin but compete with the macrolide carbomycin, which is a peptidyl transferase inhibitor. We infer from these and previous results that tiamulin and valnemulin interact with the rRNA in the peptidyl transferase slot on the ribosomes in which they prevent the correct positioning of the CCA-ends of tRNAs for peptide transfer.

Anti-Bacterial Agents↗
PubMed2003

A 49 kDa microtubule cross-linking protein from Artemia franciscana is a coenzyme A-transferase.

Embryos and larvae of the brine shrimp, Artemia franciscana, were shown previously to possess a protein, now termed p49, which cross-links microtubules in vitro. Molecular characteristics of p49 were described, but the protein's identity and its role in the cell were not determined. Degenerate oligonucleotide primers designed on the basis of peptide sequence obtained by Edman degradation during this study were used to generate p49 cDNAs by RT-PCR and these were cloned and sequenced. Comparison with archived sequences revealed that the deduced amino acid sequence of p49 resembled the Drosophila gene product CG7920, as well as related proteins encoded in the genomes of Anopheles and Caenorhabditis. Similar proteins exist in several bacteria but no evident homologues were found in vertebrates and plants, and only very distant homologues resided in yeast. When evolutionary relationships were compared, p49 and the homologues from Drosophila, Anopheles and Caenorhabditis formed a distinct subcluster within phylogenetic trees. Additionally, the predicted secondary structures of p49, 4-hydroxybutyrate CoA-transferase from Clostridium aminobutyricum and glutaconate CoA-transferase from Acidaminococcus fermentans were similar and the enzymes may possess related catalytic mechanisms. The purified Artemia protein exhibited 4-hydroxybutyrate CoA-transferase activity, thereby establishing p49 as the first crustacean CoA-transferase to be characterized. Probing of Western blots with an antibody against p49 revealed a cross-reactive protein in Drosophila that associated with microtubules, but to a lesser extent than did p49 from Artemia.

Adenosine Triphosphate↗
PubMed1999

Characterization of the ternary complex between Rab7, REP-1 and Rab geranylgeranyl transferase.

Geranylgeranylation is a post-translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein (REP) and are presented to the Rab geranylgeranyl transferase (RabGGTase) which covalentely modifies the Rab protein with two geranylgeranyl moieties. After prenylation, the Rab protein remains in complex with REP and is delivered to the target membrane by the latter. In this work, we show that RabGGTase can form a stable complex with Rab7-REP in the absence of its lipid substrate geranylgeranyl pyrophosphate. In order to characterize this interaction, we developed three fluorescence assays reporting on the interaction of RabGGTase with the Rab7-REP complex. For this interaction we determined a Kd value of about 120 nM. Association of RabGGTase with the Rab7-REP complex occurs with a rate constant of approximately 108 M-1 x s-1. We demonstrate that the state of the nucleotide bound to Rab7 does not influence the affinity of RabGGTase for the Rab7-REP-1 complex. Finally, we address the issue of substrate specificity of RabGGTase. Titration experiments demonstrate that, in contrast with farnesyl transferase, RabGGTase does not recognize a defined C-terminal sequence motif. Experiments using Rab7 mutants in which the last 16 amino acids were either mutated or truncated revealed that the distal part of the C-terminus makes only a limited contribution to the binding affinity between RabGGTase and the Rab7-REP-1 complex. This demonstrates the functional dissimilarity between RabGGTase and geranylgeranyl transferase I and farnesyl transferase, which interact specifically with the C-terminus of their substrates. Based on these experiments, we propose that RabGGTase recognizes the overall structure arising from the association of Rab and REP and then 'scans' the flexible C-terminus to position the proximal cysteines into the active site.

Alkyl and Aryl Transferases↗