scholarly journals Characterization of methylation of rat liver cytosolic glutathione S-transferases by using reverse-phase h.p.l.c. and chromatofocusing

1990 ◽  
Vol 270 (2) ◽  
pp. 483-489 ◽  
Author(s):  
J A Johnson ◽  
T L Neal ◽  
J H Collins ◽  
F L Siegel
Keyword(s):  
Rat Liver ◽  
Time Course ◽  
Glutathione S Transferase ◽  
Reverse Phase ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases ◽  
Rat Liver Cytosol ◽  
Gst Activity

Glutathione S-transferase (GST) subunits in rat liver cytosol were separated by reverse-phase h.p.l.c.; five major proteins were isolated and identified as subunits 1, 2, 3, 4 and 8. F.p.l.c. chromatofocusing resolved the affinity-purified GST pool into nine different isoenzymes. The five basic (Alpha class) dimeric peaks of GST activity were 1-1, 1-2a, 1-2b, 2-2a and 2-2b. Reverse-phase h.p.l.c. analysis revealed that subunit 8 was also present in the protein peaks designated 1-1, 1-2a and 1-2b. The four neutral (Mu class) isoenzymes were 3-3, 3-4, 3-6 and 4-4. The GST pool was methylated in vitro before reverse-phase h.p.l.c. or f.p.l.c. chromatofocusing. Chromatofocusing indicated that the Mu class isoforms (3-3, 3-4 and 4-4) were the primary GSTs methylated, and h.p.l.c. analysis confirmed that subunits 3 and 4 were the major methyl-accepting GST subunits. The addition of calmodulin stimulated the methylation in vitro of GST isoenzymes 3-3, 3-4 and 4-4 by 3.0-, 7.5- and 9.9-fold respectively. Reverse-phase h.p.l.c. also indicated that only the methylation of GST subunits 3 and 4 was stimulated by calmodulin. Basic GST isoenzymes were minimally methylated and the methylation was not enhanced by calmodulin. Investigation of the time course of methylation of GST subunits 3 and 4 indicated that at incubation times less than 4 h the methylation of both Mu class subunits was stimulated by calmodulin, and that under such conditions subunit 4 was the preferred substrate. In contrast, there was essentially no calmodulin-stimulated methylation at incubation times of 4 or 6 h, and the methylation of subunit 3 was predominant. Kinetic parameters at 2 h of incubation were determined in the presence and in the absence of calmodulin. The addition of calmodulin doubled the Vmax. for methylation of both subunits 3 and 4 and decreased the Km of subunit 4 for S-adenosyl-L-methionine 3.6-fold. Finally, methylation was substoichiometric and after 6 h of incubation ranged from 2.8 to 7.6% on a mole-to-mole basis for subunits 4 and 3 respectively.

Cytosolic glutathione S-transferases of Oesophagostomum dentatum

Parasitology ◽  
2008 ◽  
Vol 135 (10) ◽  
pp. 1215-1223 ◽  
Author(s):  
A. JOACHIM ◽  
B. RUTTKOWSKI
Keyword(s):  
Amino Acids ◽  
Pcr Primers ◽  
Mrna Levels ◽  
Fourth Stage ◽  
Glutathione S Transferase ◽  
Cdna Sequences ◽  
Oesophagostomum Dentatum ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases

SUMMARYOesophagostomum dentatum stages were investigated for glutathione S-transferase (GST) expression at the protein and mRNA levels. GST activity was detected in all stages (infectious and parasitic stages including third- and fourth-stage larvae of different ages as well as males and females) and could be dose-dependently inhibited with sulfobromophthalein (SBP). Addition of SBP to in vitro larval cultures reversibly inhibited development from third- to fourth-stage larvae. Two glutathione-affinity purified proteins (23 and 25 kDa) were detected in lysates of exsheathed third-stage larvae by SDS-PAGE. PCR-primers were designed based on peptide sequences and conserved GST sequences of other nematodes for complete cDNA sequences (621 and 624 nt) of 2 isoforms, Od-GST1 and Od-GST2, with 72% nucleotide similarity and 75% for the deduced proteins. Genomic sequences consisted of 7 exons and 6 introns spanning 1296 bp for Od-GST1 and 1579 and 1606 bp for Od-GST2. Quantitative real-time-PCR revealed considerably elevated levels of Od-GST1 in the early parasitic stages and slightly reduced levels of Od-GST2 in male worms. Both Od-GSTs were most similar to GST of Ancylostoma caninum (nucleotides: 73 and 70%; amino acids: 80 and 73%). The first three exons (75 amino acids) corresponded to a synthetic prostaglandin D2 synthase (53% similarity). O. dentatum GSTs might be involved in intrinsic metabolic pathways which could play a role both in nematode physiology and in host-parasite interactions.

scholarly journals Purification and characterization of a new cytosolic glutathione S-transferase (glutathione S-transferase X) from rat liver

1983 ◽  
Vol 215 (3) ◽  
pp. 617-625 ◽  
Author(s):  
T Friedberg ◽  
U Milbert ◽  
P Bentley ◽  
T M Guenther ◽  
F Oesch
Keyword(s):  
Rat Liver ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Glutathione S Transferase ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases

A hitherto unknown cytosolic glutathione S-transferase from rat liver was discovered and a method developed for its purification to apparent homogeneity. This enzyme had several properties that distinguished it from other glutathione S-transferases, and it was named glutathione S-transferase X. The purification procedure involved DEAE-cellulose chromatography, (NH4)2SO4 precipitation, affinity chromatography on Sepharose 4B to which glutathione was coupled and CM-cellulose chromatography, and allowed the isolation of glutathione S-transferases X, A, B and C in relatively large quantities suitable for the investigation of the toxicological role of these enzymes. Like glutathione S-transferase M, but unlike glutathione S-transferases AA, A, B, C, D and E, glutathione S-transferase X was retained on DEAE-cellulose. The end product, which was purified from rat liver 20 000 g supernatant about 50-fold, as determined with 1-chloro-2,4-dinitrobenzene as substrate and about 90-fold with the 1,2-dichloro-4-nitrobenzene as substrate, was judged to be homogeneous by several criteria, including sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, isoelectric focusing and immunoelectrophoresis. Results from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration indicated that transferase X was a dimer with Mr about 45 000 composed of subunits with Mr 23 500. The isoelectric point of glutathione S-transferase X was 6.9, which is different from those of most of the other glutathione S-transferases (AA, A, B and C). The amino acid composition of transferase X was similar to that of transferase C. Immunoelectrophoresis of glutathione S-transferases A, C and X and precipitation of various combinations of these antigens by antisera raised against glutathione S-transferase X or C revealed that the glutathione S-transferases A, C and X have different electrophoretic mobilities, and indicated that transferase X is immunologically similar to transferase C, less similar to transferase A and not cross-reactive to transferases B and E. In contrast with transferases B and AA, glutathione S-transferase X did not bind cholic acid, which, together with the determination of the Mr, shows that it does not possess subunits Ya or Yc. Glutathione S-transferase X did not catalyse the reaction of menaphthyl sulphate with glutathione, and was in this respect dissimilar to glutathione S-transferase M; however, it conjugated 1,2-dichloro-4-nitrobenzene very rapidly, in contrast with transferases AA, B, D and E, which were nearly inactive towards that substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Rat kidney glutathione S-transferase 1 subunits have C-terminal truncations

1996 ◽  
Vol 314 (3) ◽  
pp. 1017-1025 ◽  
Author(s):  
Horng-I. YEH ◽  
Jing-Yu LEE ◽  
Shu-Ping TSAI ◽  
Cheng-Hsilin HSIEH ◽  
Ming F. TAM
Keyword(s):  
Rat Liver ◽  
Peptide Mapping ◽  
Rat Kidney ◽  
Cdna Sequencing ◽  
Sequencing Data ◽  
Glutathione S Transferase ◽  
Reverse Phase Hplc ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases ◽  
Molecular Masses

Cytosolic glutathione S-transferases (GSTs) from rat kidneys were purified by a combination of glutathione and S-hexylglutathione affinity columns. The isolated GSTs were subjected to reverse-phase HPLC and electrospray MS analysis. The major GST isoenzymes expressed in kidney are subunits 1, 2, 7 and 8. GST 1´, 3, and 4 are expressed in minor amounts. GST 10 is barely detectable in the male kidney cytosol. The molecular masses of these rat kidney GST subunits were determined by MS. The values obtained for subunits 1´, 2, 3, 4, 7, 8 and 10 are identical with those obtained for rat liver GSTs. Rat kidney GST 1 consists of three polypeptides, with molecular masses of 25517, 25372 and 24982 Da. Results from peptide mapping, MS and amino-acid-sequencing analyses indicate that the major components were generated by deleting the C-terminal phenylalanine (24982 Da) and the C-terminal IFKF tetrapeptide (25372 Da) from the GST 1 subunit, respectively. The 1-chloro-2,4-dinitrobenzene-conjugating and peroxidase activities of kidney GST 1 are substantially lower than for its counterpart from rat liver. In addition, rat kidney GST 1 has an arginine and a valine residue at positions 151 and 207 respectively. The results are in contradiction with the SWISS-PROT and GenBank rat liver GST 1 cDNA-sequencing data, which give a lysine and a methionine at the corresponding positions. Further analyses indicate that rat liver GST 1 also has C-terminal phenylalanine deletion, and an arginine and a valine residue at positions 151 and 207 respectively. However, the C-terminal-tetrapeptide-deleted form was not observed for rat liver GST 1.

scholarly journals The characterization of glutathione S-transferases from rat olfactory epithelium

1993 ◽  
Vol 290 (1) ◽  
pp. 199-204 ◽  
Author(s):  
K K Banger ◽  
E A Lock ◽  
C J Reed
Keyword(s):  
Substrate Specificity ◽  
Olfactory Epithelium ◽  
Liver Microsomes ◽  
Subunit Composition ◽  
Reverse Phase ◽  
Glutathione S Transferases ◽  
Gst Activity

The glutathione S-transferases (GSTs) of rat olfactory epithelium have been characterized with regard to substrate specificity and subunit composition and compared to those of the liver. The presence of cytosolic GST activity in rat olfactory epithelium was confirmed and, using 1-chloro-2,4-dinitrobenzene as substrate, was found to be approximately one-third that of the liver. Olfactory microsomal GST activity was greater than that of liver microsomes and could be activated by treatment with the sulphydryl agent N-ethylmaleimide. The subunit and isoenzyme profile of GSTs in the olfactory epithelium was investigated using a number of techniques. (1) Olfactory GSTs were characterized using a range of relatively subunit-specific substrates. Activities ranged from 40-90% of those found in liver. Most noticeable was the extremely low olfactory activity with the substrate specific for subunit 1. (2) Immunoblotting with antibodies against specific rat hepatic GSTs confirmed the presence of a number of subunits and the absence of subunit 1. (3) F.p.l.c. chromatofocusing and reverse-phase h.p.l.c. indicated that the cytosolic GST profile of olfactory epithelium is unique and is made up of subunits 2, 3, 4, 7, 8 and 11 with subunits 3 and 4 predominating. There is an absence of isoenzymes containing subunit 1.

Antibiotics as Inhibitor of Glutathione S-transferase: Biological Evaluation and Molecular Structure Studies

2021 ◽  
Vol 22 ◽  
Author(s):  
Adnan Ayna ◽  
Luqman Khosnaw ◽  
Yusuf Temel ◽  
Mehmet Ciftci
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
Biological Evaluation ◽  
Glutathione S Transferase ◽  
Scopolamine Butylbromide ◽  
Glutathione S Transferases ◽  
Concentration Graphs ◽  
Compound Concentration

Background: The glutathione S-transferases (GSTs) are family of enzymes that are notable for their role in phase II detoxification reactions. Antibiotics have been reported to have several adverse effects on the activity of the enzymes in mammals. Aim: The aim of this study was structural and biochemical characterization of rat erythrocyte GST and understanding the effects of gentamicin, clindamycin, cefazolin, ampicillin and scopolamine butylbromide on the activity of human erythrocyte GST using rat as a model. Methods: The enzyme was purified by GSH-agarose affinity chromatography. In vitro GST enzyme activity was measured at 25°C using CDNB as a model substrate. IC50 of drugs were measured by activity %–vs compound concentration graphs. Lineweaver–Burk graphs were drawn to determine the inhibition type and Ki constants for the drugs. The structure of the enzyme was predicted via Protein Homology/analogY Recognition Engine. Results: In this study, GST was purified from rat erythrocyte with a specific activity of 6.3 EU/mg protein, 44 % yield and 115 fold. Gentamicin and clindamycin inhibited the enzymatic activity with IC50 of 1.69 and 6.9 mM and Ki of 1.70 and 2.36 mM, respectively. Ampicillin and scopolamine butylbromide were activator of the enzyme while the activity of the enzyme was insensitive to cefazolin. The enzyme was further characterized by homology modeling and sequence alignment revealing similarities with human GST. Conclusion: Collectively, it could be concluded that gentamicin and clindamycin are the inhibitors of erythrocyte GST.

scholarly journals 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

1981 ◽  
Vol 197 (2) ◽  
pp. 491-502 ◽  
Author(s):  
J D Hayes ◽  
R C Strange ◽  
I W Percy-Robb
Keyword(s):  
Rat Liver ◽  
Lithocholic Acid ◽  
Binding Activity ◽  
Glutathione S Transferase ◽  
Liver Cytosol ◽  
Molecular Weights ◽  
Tryptic Digest ◽  
Glutathione S Transferases ◽  
Limited Digestion ◽  
Rat Liver Cytosol

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.

scholarly journals Two immunologically distinct Yc-type subunits are present in rat lung glutathione S-transferases

1984 ◽  
Vol 224 (1) ◽  
pp. 335-338 ◽  
Author(s):  
S V Singh ◽  
Y C Awasthi
Keyword(s):  
Glutathione Peroxidase ◽  
Rat Liver ◽  
Peroxidase Activity ◽  
Gel Electrophoresis ◽  
Rat Lung ◽  
Glutathione Peroxidase Activity ◽  
Glutathione S Transferase ◽  
Two Dimensional Gel Electrophoresis ◽  
Glutathione S Transferases

Two types of 25 000-Mr subunits are present in rat lung glutathione S-transferase I (pI 8.8). These subunits, designated Yc and Yc', are immunologically and functionally distinct from each other. The homodimers YcYc (pI 10.4) and Yc'Yc' (pI 7.6) obtained by hybridization in vitro of the two subunits of glutathione S-transferase I (pI 8.8) were isolated and characterized. Results of these studies indicate that only the Yc subunits express glutathione peroxidase activity and cross-react with the antibodies raised against glutathione S-transferase B (YaYc) or rat liver. The Yc' subunits do not express glutathione peroxidase activity and do not cross-react with the antibodies raised against glutathione S-transferase B of rat liver. The amino acid compositions of these two subunits are also different. These two subunits can also be separated by the two-dimensional gel electrophoresis of glutathione S-transferase I (pI 8.8) of rat lung.

scholarly journals BENZYLIDENE CYCLOPENTANONE DERIVATIVES AS INHIBITORS OF RAT LIVER GLUTATHIONE S-TRANSFERASE ACTIVITIES

2010 ◽  
Vol 5 (1) ◽  
pp. 71-75 ◽  
Author(s):  
Sudibyo Martono
Keyword(s):  
Rat Liver ◽  
Potent Inhibitor ◽  
Inflammatory Activity ◽  
Glutathione S Transferase ◽  
Inhibitory Potency ◽  
Liver Cytosol ◽  
Curcumin Analogues ◽  
Glutathione S Transferases ◽  
Anti Inflammatory ◽  
Rat Liver Cytosol

The effect of the curcumin analogues, 2,6-bis-(4-hydroxy-3-methoxy benzylidene) cyclopentanone (B1) and two of its derivatives on m class glutathione S-transferases (GSTs) from phenobarbital-induced and uninduced rat liver cytosol has been studied to elucidate their anti-inflammatory activity. GST activity was monitored spectrophotometrically using 1,2-dichloro-4-nitrobenzene. B1 was the most potent inhibitor of GSTs, both in uninduced and in phenobarbital-induced rat liver cytosol. These inhibitory properties might be explained as part of the anti-inflammatory activity of benzylidene cyclopentanone derivatives (B1 and B12).   Keywords: curcumin; benzylidene cyclopentanone; inhibitory potency; glutathione S-transferases mesoporous

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