Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To set up an in vitro amidating system, a recombinant human calcitonin with a glycine at C termial (mhCT-Gly) was used as the amidating substrate of recombinant rat peptidylglycine alpha-amidating monooxygenase (rPAM). First, the mhCT-Gly gene was synthesized and cloned into a fusion expression vector to get an expression plasmid pGEXCT. The GST-fused mhCT-Gly was highly expressed in E.coli BL21(DE3) harboring the pGEXCT, and was purified rapidly by affinity chromatography. Second, using the method of ultrafiltration, the rPAM was prepared from the supernatant of cultured transfectant CHO cells which express rPAM stably. Finally, the in vitro amidating experiments were carried out using GST-mhCT-Gly as substrate and the prepared rPAM. The results of dot blot with the specific antibody and of mass spectrum assay indicated that amidating product GST-hCT-NH(2) could be easily detected. This study provides a useful method for the amidation of recombinant products in vitro.
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PMID:In Vitro Amidating Processing of Products Expressed by Gene Engineering. 1207 61

Rapid modulation of the surface number of certain ionotropic receptors is achieved by altering the relative rates of insertion and internalization. These receptors are internalized by a clathrin-mediated pathway; however, a motif that is necessary for endocytosis of ionotropic receptors has not yet been identified. Here, we identified a motif that is required for constitutive and agonist-regulated internalization of the ionotropic P2X(4) receptor. Three amino acids in the C terminus of P2X(4) (Tyr(378), Gly(381), and Leu(382)) compose a non-canonical tyrosine-based sorting signal of the form YXXGL. We found that P2X(4) protein was present in clathrin-coated vesicles isolated from rat brain and that a glutathione S-transferase fusion of the P2X(4) C terminus pulled down the adaptor protein-2 complex from brain extract. Mutation of either the tyrosine-binding pocket of the mu2 subunit of adaptor protein-2 or the YXXGL motif in the receptor C terminus caused a decrease in receptor internalization and a dramatic increase in the surface expression of P2X(4) receptors. The YXXGL motif represents a non-canonical tyrosine-based sorting signal that is necessary for efficient endocytosis of the P2X(4) receptor. Similar motifs are present in other receptors and may be important for the control of their functional expression.
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PMID:Identification of a non-canonical tyrosine-based endocytic motif in an ionotropic receptor. 1210 1

The presence of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) paradoxically increases the mutagenicity and cytotoxicity of 1,2-dibromoethane (DBE) in Escherichia coli. This enhancement of genotoxicity did not occur when the inactive C145A mutant of human AGT (hAGT) was used. Also, hAGT did not enhance the genotoxicity of S-(2-haloethyl)glutathiones that mimic the reactive product of the reaction of DBE with glutathione, which is catalyzed by glutathione S-transferase. These experiments support a mechanism by which hAGT activates DBE. Studies in vitro showed a direct reaction between purified recombinant hAGT and DBE resulting in a loss of AGT repair activity and a formation of an hAGT-DBE conjugate at Cys(145). A 2-hydroxyethyl adduct was found by mass spectrometry to be present in the Gly(136)-Arg(147) peptide from tryptic digests of AGT reacted with DBE. Incubation of AGT with DBE and oligodeoxyribonucleotides led to the formation of covalent AGT-oligonucleotide complexes. These results indicate that DBE reacts at the active site of AGT to generate an S-(2-bromoethyl) intermediate, which forms a highly reactive half-mustard at Cys(145). In the presence of DNA, the DNA-binding function of AGT facilitates formation of DNA adducts. In the absence of DNA, the intermediate undergoes hydrolytic decomposition to form AGT-Cys(145)-SCH(2)CH(2)OH.
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PMID:Paradoxical enhancement of the toxicity of 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase. 1215 4

The activation of factor X (fX) to factor Xa (fXa) marks the penultimate step in the coagulation cascade and modulating fXa activity may be effective for antithrombotic therapy. Even though fXa inhibitors are screened using in vitro inhibition of human fXa (HfXa) while subsequent evaluation uses in vivo rabbit models, there is limited knowledge of species differences between the coagulation proteins. When comparing amino acid sequences for the human (HfX) and rabbit (RafX) protein, differences are found in the activation peptide and active site regions. In order to study the relative functional characteristics of HfX and RafX, we asked (1) whether fX from the two species is immunologically related, (2) whether the two proteins are activated to fXa in a similar manner, (3) whether HfXa and rabbit factor Xa (RafXa) have similar catalytic activities toward tripeptide substrates. To answer (1), we expressed RafX-glutathione S-transferase (RafX-GST) fusion protein in bacteria and purified the protein for use as an antigen. The resulting monoclonal antibodies were suitable for affinity purification of plasma RafX and for effective anticoagulation in rabbit plasma clotting assays. We found two antibodies (mAb 214 and mAb 290) that anticoagulated rabbit plasma in a dose responsive manner but did not cross-react with human plasma. At a concentration of 500 nM, mAb 214 attained a two-fold extension of rabbit plasma activated partial thromboplastin time (aPTT). To answer (2), we purified plasma RafX and compared the activation of HfX and RafX with Russell's viper venom (RVV-X). Under equivalent reaction conditions, conversion was 30% slower for the rabbit protein. To answer (3), amidolytic activity of HfXa and RafXa were assayed by cleavage of three para-nitroanilide (pNA) substrates (S2222 [Bz-Ile-Glu(gamma-OR)-Gly-Arg-pNA.HCl], S2765 [Z-D-Arg-Gly-Arg-pNA.HCl] and Spectrozyme Xa [MeO-CO-D-CHG-Gly-Arg-pNA.AcOH]). Michaelis constants (K(m)) for the rabbit protein were 187, 72 and 69 microM, respectively, and for the human analog, 255, 63 and 135 microM, respectively. Comparing the extent of substrate turnover (V(max)) for HfXa and RafXa, the latter was shown to cleave all three substrates at a reduced rate. Based on these observations, it can be speculated that the relative antithrombotic potency of active site directed fXa inhibitors might be different between the two species. Predicted human therapeutic doses derived from in vivo results in rabbit models should therefore take species variation into consideration.
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PMID:Differences between human and rabbit coagulation factor X-implications for in vivo models of thrombosis. 1216 92

Using anti-GST-UVS.2 antibody and vitellin envelope (VE) as probes, the Xenopus laevis hatching enzyme (HE) was purified about 90-fold over the starting crude HE by gel-filtration and ionexchange chromatography, and its enzymatic and biochemical properties were studied. The HE has a molecular weight of 60 kD, and has high proteolytic and VE-solubilizing activities. It was very unstable during purification, and was digested easily into a 40 kD molecule, which had no VE-solubilizing activity, but still retained its proteolytic activity. The 40 kD molecule probably represents only the main protease domain in the 60 kD molecule, with two CUB repeats lost. The results on its sensitivity to EDTA and some other metal ions, combined with the occurrence of the astacin family metalloprotease-specific "HExHxxGFxHE" sequence in the deduced HE amino acid sequence, indicate that the HE is a metalloprotease. HE is very sensitive to trypsin-specific inhibitors such as leupeptin, p-APMSF, SBTI, LBTI, ovomucoid, bestatin, DFP and TLCK, which indicates that it is a trypsin-type protease. Boc-Leu-Gly-Arg-MCA had been determined to be its specific MCA-substrate.
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PMID:Purification and Biochemical Characterization of the Hatching Enzyme from Xenopus laevis. 1217 2

The leader proteinase (L(pro)) of foot and mouth disease virus is a papain-like cysteine proteinase. After processing itself from the polyprotein, L(pro) then cleaves the host protein eukaryotic initiation factor (eIf) 4GI, thus preventing protein synthesis from capped mRNA in the infected cell. We have investigated L(pro) interaction with eIF4GI and its isoform, eIF4GII. L(pro), expressed as a catalytically inactive fusion protein with glutathione S-transferase, binds specifically to eIF4G isomers in rabbit reticulocyte lysates. Deletion and specific mutagenesis were used to map the binding domain on L(pro) to residues 183-195 of the C-terminal extension and to residue Cys(133). These residues of the C-terminal extension and Cys(133) are adjacent in the crystal structure but lie about 25 A from the active site. The region on eIF4GI recognized by the L(pro) C-terminal extension was mapped to residues 640-669 using eIF4GI fragments generated by proteolysis or by in vitro translation. The L(pro) cleavage site at Gly(674) downward arrow Arg(675) was not necessary for binding. Similar experiments with human rhinovirus 2A proteinase (2A(pro)), a chymotrypsin-like cysteine proteinase that also cleaves eIF4G isoforms, revealed that 2A(pro) can also bind to eIF4GI fragments lacking its cleavage site. These experiments strongly suggest a novel interaction between picornaviral proteinases and eIF4G isoforms.
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PMID:Recognition of eukaryotic initiation factor 4G isoforms by picornaviral proteinases. 1222 54

In human glutathione transferase P1-1 (hGSTP1-1) position 146 is occupied by a glycine residue, which is located in a bend of a long loop that together with the alpha6-helix forms a substructure (GST motif II) maintained in all soluble GSTs. In the present study G146A and G146V mutants were generated by site-directed mutagenesis in order to investigate the function played by this conserved residue in folding and stability of hGSTP1-1. Crystallographic analysis of the G146V variant, expressed at the permissive temperature of 25 degrees C, indicates that the mutation causes a substantial change of the backbone conformation because of steric hindrance. Stability measurements indicate that this mutant is inactivated at a temperature as low as 32 degrees C. The structure of the G146A mutant is identical to that of the wild type with the mutated residue having main-chain bond angles in a high energy region of the Ramachandran plot. However even this Gly --> Ala substitution inactivates the enzyme at 37 degrees C. Thermodynamic analysis of all variants confirms, together with previous findings, the critical role played by GST motif II for overall protein stability. Analysis of reactivation in vitro indicates that any mutation of Gly-146 alters the folding pathway by favoring aggregation at 37 degrees C. It is hypothesized that the GST motif II is involved in the nucleation mechanism of the protein and that the substitution of Gly-146 alters this transient substructure. Gly-146 is part of the buried local sequence GXXh(T/S)XXDh (X is any residue and h is a hydrophobic residue), conserved in all GSTs and related proteins that seems to behave as a characteristic structural module important for protein folding and stability.
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PMID:Contribution of glycine 146 to a conserved folding module affecting stability and refolding of human glutathione transferase p1-1. 1241 96

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand (PEF) protein family. ALG-2 forms a homodimer, a heterodimer with another PEF protein, peflin, and a complex with its interacting protein, named Alix or AIP1. We previously identified annexin XI as a novel ALG-2-binding partner. Both the N-terminal regulatory domain of annexin XI (Anx11N) and the ALG-2-binding domain of Alix/AIP1 are rich in Pro, Gly, Ala, Tyr and Gln. This PGAYQ-biased amino acid composition is also found in the N-terminal extension of annexin VII (Anx7N). Using recombinant ALG-2 proteins and the glutathione S-transferase (GST) fusion proteins of Anx7N and Anx11N, the direct Ca(2+)-dependent interaction was analyzed by a biotin-tagged ALG-2 overlay assay and by a real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. Both GST-Anx7N and GST-Anx11N showed similar binding kinetics against ALG-2 as well as ALG-2-DeltaN23, which lacked the hydrophobic N-terminal region. Two binding sites were predicted in both Anx7N and Anx11N, and the dissociation constants (K(d)) were estimated to be approximately 40-60 nM for the high-affinity site and 500-700 nM for the low-affinity site.
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PMID:The penta-EF-hand domain of ALG-2 interacts with amino-terminal domains of both annexin VII and annexin XI in a Ca2+-dependent manner. 1244 60

NF-kappaB1 p105 functions both as a precursor of NF-kappaB1 p50 and as a cytoplasmic inhibitor of NF-kappaB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-alpha), the IkappaB kinase (IKK) complex rapidly phosphorylates NF-kappaB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-alpha to trigger p105 degradation, which releases the associated Rel/NF-kappaB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser(927)-Gly-Val-Glu-Thr-Ser(932)) homologous to the IKK target sequence in IkappaBalpha. In this study, TNF-alpha-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1(-/-) and IKK2(-/-) double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-alpha to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105(758-967) fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for betaTrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of betaTrCP by RNA interference blocks TNF-alpha-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that betaTrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, betaTrCP affinity for the IKK-phosphorylated sequence on p105 is substantially lower than that on IkappaBalpha. Thus, it appears that reduced p105 recruitment of betaTrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-alpha stimulation relative to that for IkappaBalpha.
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PMID:betaTrCP-mediated proteolysis of NF-kappaB1 p105 requires phosphorylation of p105 serines 927 and 932. 1248 91

To investigate the effect of GST-KGDX (glutathione S-transferase-Lys-Gly-Asp-X) fusion protein, GP IIb/IIIa receptor antagonist, on platelet function in vitro. The KGDX (Lys-Gly-Asp-X) gene was assembled from 2 synthetic oligonucleotides, 36 bp in length, using BamH I and Xho I restriction enzyme sites at the end of the gene for cloning into the expression vector pGEX4T-1. Expression of fusion protein was directed by the tac promoter. The Escherichia coli DH5a contained the plasmid pGEX-4T-1-KGDX was expressed by 37 degrees C heat induction. The fusion protein of KGDX with glutathione S-transferase (GST-KGDX) was purified in one step from the bacterial lysate by glutathione-agarose beads for affinity chromatography. GST-KGDX was found to be soluble and abundant, the yield of 35 mg/L of cultures was obtained. The GST-KGDX was expressed in E. coli to a level of 48.02% of total cellular protein. GST-KGDX inhibited ADP-induced human platelet aggregation stronger than GST (P < 0.05 or < 0.01). In flow cytometry assay for fibrinogen binding, both GST and GST-KGDX inhibited platelet aggregation by binding with high affinity to GPIIb/IIIa. Mean fluorescence intensity of GST-KGDX fusion protein was significantly higher than that of GST. It is concluded that the GST-KGDX fusion protein can be produced by E. coli and used as an antiplatelet agent.
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PMID:[Expression and purification of recombinant glycoprotein (GP) IIb/IIIa receptor antagonists]. 1251 16


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