Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutaredoxin catalyzes glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH and glutathione reductase and has an active site disulfide/dithiol with the sequence -Cys-Pro-Tyr-Cys-. Calf thymus glutaredoxin (thioltransferase), which contains two additional structural half-cystine residues, was purified to homogeneity, using a modification of the previously described isolation procedure. This method involved a pI-shift of glutaredoxin, obtained after oxidation of the fully reduced form with hydroxyethyl-disulfide, followed by CM-Sepharose chromatography. On both SDS- and IEF-gels the protein migrated as one band (M(r) 12,000). The pure protein was used to affinity-purify rabbit antiglutaredoxin antibodies obtained by immunization with the oxidized form of glutaredoxin. Using these antibodies the distribution of glutaredoxin was mapped in calf organs and tissues by Western blots and by immunohistochemistry. Glutaredoxin was demonstrated in all organs investigated. Western blots showed the presence of weak additional high molecular weight bands of unknown identity in certain organs. The immunohistochemical analyses revealed that glutaredoxin is highly expressed in a wide variety of cell types, both epithelial and mesenchymal. The distribution and occurrence in the calf organs was similar to that previously described for thioredoxin in the rat. There were some exceptions: e.g., follicular cells in the ovary did not contain immunohistochemically demonstrable glutaredoxin but expressed thioredoxin. Particularly striking were observations of strong glutaredoxin immunoreactivity in oocytes in the ovary and the pattern of glutaredoxin in epithelial tissue of the skin and tongue reflecting differential expression during cell differentiation. The distribution demonstrated that glutaredoxin serves functions apart from the originally described role as hydrogen donor for ribonucleotide reductase which only occurs in replicating cells. Such functions should relate particularly to glutathione-catalyzed protein disulfide oxidoreductions and cellular signalling by redox regulating mechanisms.
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PMID:Immunochemical characterization and tissue distribution of glutaredoxin (thioltransferase) from calf. 792 87

The genomic DNA for the two Drosophila genes, gstD1 and gstD21, were engineered for expression in Escherichia coli by polymerase chain reaction using a pair of specially designed primers. This newly designed expression system produced consistently high yields of the recombinant glutathione S-transferases (GSTs), which were purified to electrophoretic homogeneity by S-hexyl-GSH affinity chromatography. Consistent with their differences in size, GST D1 and GST D21 displayed different mobilities on SDS-polyacrylamide gel electrophoresis. Circular dichroism spectrometry revealed some differences in the protein secondary structural organization between the two GST D isozymes. Polyclonal antibodies against GST D1 and GST D21 revealed that they are immunologically distinct from each other. The GST D1 antiserum cross-reacted weakly with GST D21, but the GST D21 antiserum had no detectable cross-reactivity with GST D1. The amino acid sequences of GST D1 and GST D21 have 70% identity. GST D1 is active toward CDNB with 17% of the catalytic efficiency of the human alpha GST121, whereas CDNB is a poor substrate for GST D21. Both GST D1 and GST D21 have similar levels of GSH peroxidase activity against cumene hydroperoxide. Another major difference in substrate specificities between GST D1 and GST D21 is in the activity of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) dehydrochlorinase, which exists only in the GST D1 isozyme. This is the first definitive demonstration that DDT dehydrochlorinase activity is an intrinsic property of a Drosophila GST. Our results suggest that GST D1 may play a role in DDT metabolism in Drosophila.
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PMID:Biochemical characterization of Drosophila glutathione S-transferases D1 and D21. 796 18

There are three selenium-containing proteins in human plasma: glutathione peroxidase (GSH-Px-P), albumin and selenoprotein Ph, the human analogue to selenoprotein P from rat plasma. Selenoprotein Ph was separated from the two other selenium-containing proteins by Heparin Sepharose chromatography and was shown to have about 60-70% of the total plasma selenium, while both GSH-Px-P and albumin contain about 15%. A 2588-fold purification from human plasma was achieved by using a four-step procedure. SDS-PAGE of the purified selenoprotein revealed, besides one contaminant selenium-free protein band at about 70 kDa, one selenium-containing band ranging from 54 to 67 kDa with a maximum at 63 kDa. This microheterogeneity, also recognized by IEF, may be due to the glycprotein nature of the selenoprotein Ph. The determination of the molecular mass of the native protein varied from 65 kDa using gel filtration on Fraktogel HW 55 to 89 kDa on Sephacryl S-200 HR, suggesting an interaction between the gel-matrices and selenoprotein Ph.
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PMID:Purification of selenoprotein Ph from human plasma. 801 51

GSH-dependent enzymic reduction of dehydroascorbic acid to ascorbic acid has been studied in rat liver cytosol. After gel filtration of cytosol on Sephadex G-100 SF, dehydroascorbate reductase activity was recovered in two distinct peaks, one corresponding to glutaredoxin (an enzyme already known for its dehydroascorbate reductase activity) and another, much larger one, corresponding to a novel enzyme different from glutaredoxin. The latter was purified to apparent homogeneity. The purification process involved (NH4)2SO4 fractionation, followed by DEAE-Sepharose, Sephadex G-100 SF and Reactive Red chromatography. SDS/PAGE of the purified enzyme in either the presence or absence of 2-mercaptoethanol demonstrated a single protein band of M(r) 31,000. The M(r) determined by both Sephadex G-100 SF chromatography and h.p.l.c. was found to be approx. 48,000. H.p.l.c. of the denatured enzyme gave an M(r) value identical with that obtained by SDS/PAGE (31,000). The apparent Km for dehydroascorbate was 245 microM and the Vmax. was 1.9 mumol/min per mg of protein; for GSH they were 2.8 mM and 4.5 mumol/min per mg of protein respectively. The optimal pH range was 7.5-8.0. Microsequence analysis of the electro-transferred enzyme band showed that the N-terminus is blocked. Data on internal primary structure were obtained from CNBr-and N-chlorosuccinimide-derived fragments. No significative sequence similarity was found to any of the protein sequences contained in the Protein Identification Resource database.
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PMID:Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver. 804 91

Glutathione (GSH) synthetase (EC 6.3.2.3) was purified from the fission yeast Schizosaccharomyces pombe L972h- and from the GSH synthetase deficient mutant MN101/pYS41, which harbors a plasmid containing the GSH synthetase gene of the fission yeast. GSH synthetase is expressed at 10 times higher the amount in MN101/pYS41 than in wild-type L972h-. The purified enzyme gave a single band on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (native PAGE). The molecular weight of this enzyme was determined to be 1.2 x 10(5) by Sepharose CL-6B gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that this enzyme was composed of two kinds of subunits, A (M(r) = 33 x 10(3)) and B (M(r) = 26 x 10(3)), and existed as a heterotetramer (A2B2). The enzyme purified from the wild-type fission yeast, which did not harbor the plasmid, showed the same electrophoretic mobilities on both native PAGE and SDS-PAGE and similar catalytic properties under standard conditions. This enzyme is most active at 45 degrees C and pH 8.0-8.5 with 20 mM Mg2+ + 10 mM ATP and 50 mM K+. The strict requirement for the monovalent cation is rather specific for the enzymes from yeasts. The presence of sugar components in the enzyme is also observed, similar to that in the rat kidney enzyme.
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PMID:Glutathione synthetase from the fission yeast. Purification and its unique heteromeric subunit structure. 819 97

The possible involvement of oxidative activation of liver microsomal glutathione (GSH) S-transferase by the cytochrome P450 system was investigated. When rats were given phenobarbital (PB) intraperitoneally for 3 days, liver microsomal GSH S-transferase activity was stimulated 1.3-1.4-fold and the effect of PB on the transferase was potentiated by combination with a catalase inhibitor, 3-amino-1,2,4-triazole. Immunoblotting of microsomal proteins from PB-treated rats with anti-microsomal GSH S-transferase antibody after SDS-PAGE showed the presence of a dimer of the transferase. When microsomal suspensions prepared from PB-treated rats were placed on ice without GSH, the microsomal GSH S-transferase activity gradually increased with time and reached 200% of the initial level at 3 hr when activation of the transferase by N-ethylmaleimide was lost. The time-dependent increase in GSH S-transferase activity in PB-treated microsomes was prevented by addition of 0.1 mM GSH. The increase in microsomal GSH S-transferase activity by NADPH was depressed by cytochrome P450 inhibitors such as SKF 525-A (2-diethylaminoethyl-2,2-diphenylvalerate), metyrapone or isoniazid in agreement with the concomitant decrease in generation of hydrogen peroxide in microsomes. These results indicate that the increase in GSH S-transferase activity in liver microsomes by PB treatment of rats is due to the oxidative modification of the enzyme by reactive oxygen species which are concomitantly increased following induction of cytochrome P450.
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PMID:Increase in liver microsomal glutathione S-transferase activity by phenobarbital treatment of rats. Possible involvement of oxidative activation via cytochrome P450. 825 Sep 59

The expression of glutathione transferase isoenzymes has been studied during the development of Bufo bufo embryo. By analysing the GSH-affinity purified materials in terms of substrate specificities, SDS-PAGE pattern, HPLC elution profile, we conclude that, up to stage 22, no significant changes in the expression of glutathione transferases isoenzymes occurred during Bufo bufo embryo development. At stage 25 the distribution of glutathione transferases was found to be slightly different from those of all other foregoing stages. A marked decrease of embryonic glutathione transferases subunits with a parallel appearance of new structurally and immunologically different subunits was noted in toad liver and kidney. Toad ovary continued to express embryonic glutathione transferase subunits.
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PMID:Developmental aspects of Bufo bufo embryo glutathione transferases. 835 Jun 63

We previously reported that rat glutathione transferase P-form (GST-P) is inactivated by hydrogen peroxide (H2O2). This involves formation of intra- or intersubunit disulfides, at least three extra bands with molecular masses of 21.5, 18, and 37 kDa being exhibited in addition to the native subunit band of 23.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. In the present study, GST-P mutants whose cysteine residues were independently substituted with alanine (C14A, C47A, C101A, and C169A) by site-directed mutagenesis were used to identify the cysteine residues responsible for the disulfide bond formation. C14A and C169A were much more inactivated than native GST-P by 1 mM H2O2, whereas C47A and, especially, C101A appeared insensitive to H2O2. On SDS-PAGE, the 21.5-kDa band was not detected in either C47A or C101A. Hydrogen peroxide treatment of mouse GST II, highly homologous to rat GST-P but possessing glycine instead of cysteine at the 101st residue, did not result in generation of the 21.5-kDa band and was also associated with less inactivation. This band was therefore considered to be due to an intrasubunit disulfide bond between Cys-47 and Cys-101. The 37-kDa band was suggested to be due to the formation of intersubunit disulfide bonds between Cys-47 residues in different subunits. Thus the Cys-47 residue together with Cys-101 may be located in an important region for GSH binding, disulfide bond formation between these residues resulting in steric hindrance.
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PMID:Identification of cysteine residues involved in disulfide formation in the inactivation of glutathione transferase P-form by hydrogen peroxide. 842 45

To clarify the mechanism underlying local anesthetic-induced changes in the shape of human erythrocytes from discocytes to stomatocytes, we treated erythrocytes with lidocaine, a cationic drug. Analysis of the erythrocyte membrane and cytoplasm by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the intensities of the stained bands of 62 kDa, 28 kDa and 22 kDa depended on the extent of the shape change induced by lidocaine. The change in the intensity of the 28 kDa band was particularly marked. We identified the cytoplasmic substances, i.e., the 28 kDa and 22 kDa peptides, as carbonic anhydrase (CA) and glutathione peroxidase (GSH Px)1, respectively, by immunoblotting. The 62 kDa peptide was identified as Hb by column chromatography and SDS-PAGE analysis. To identify the protein responsible for the lidocaine-induced shape change, we incorporated CA and GSH Px into ATP-MgCl2-resealed ghosts. The shape of the resealed ghosts changed upon addition of lidocaine, but only in the presence of CA. These results suggest that ATP and CA are required for the shape changes induced by lidocaine.
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PMID:Requirement of cytoplasmic components for lidocaine-induced shape change in human erythrocytes. 845 85

Glutaredoxin (Grx) (12 kDa) is a hydrogen donor for ribonucleotide reductase and also a general GSH-disulfide reductase of importance for redox regulation. To overexpress human glutaredoxin in Escherichia coli, a cDNA encoding human Grx was modified and cloned into the vector pET-3d and expressed in E. coli BL21 (DE3) by IPTG induction. High-level expression of Grx was verified by GSH-disulfide oxidoreductase activity, SDS-PAGE and immunoblotting analysis. The recombinant human Grx in its reduced form was purified to homogenity with 50% yield and exhibited the same dehydroascorbate reductase and hydrogen donor activity for ribonucleotide reductase (Km approximately 0.2 microM) as the human placenta protein. Human Grx contains a total of 5 half-cystine residues including a non-conserved Cys7 residue and is easily oxidized to form dimers during storage. A Grx mutant Cys7 to Ser was generated by site-directed mutagenesis and the protein was purified to homogeneity. The mutant protein showed full activity and exhibited a much reduced tendency to form dimers compared with the wild type protein. Peptide sequencing confirmed the mutation and removal of the N-terminal Met residue in both wild type and mutant proteins. Fluorescence spectra demonstrated only tyrosine fluorescence in human Grx with a peak at 310 nm which increased 20% upon reduction and decreased by addition of GSSG demonstrating that glutathione-containing disulfides are excellent substrates.
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PMID:High-level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys7 to Ser mutant protein. 854 5


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