Recombinant Human Glutathione Peroxidase 1, His-tagged

• Cat.No.: GPX1-1273H
• Product Overview: Recombinant human GPX1(U49C) protein, fused to His-tag at N-terminus, was expressed in E.coli and purified by using conventional chromatography techniques.

Specification

• Species: Human
• Source: Human
• Tag: His
• Description: GPX1 belongs to the glutathione peroxidase family, consisting of eight known glutathione peroxidases (Gpx1-8) in humans. GPX1 functions in the detoxification of hydrogen peroxide, and is one of the most important antioxidant enzymes in humans. The GPX1 is part of the enzymatic antioxidant defence, preventing oxidative damage to DNA, proteins and lipids by detoxifying hydrogen and lipid peroxides that may contribute to prostate cancer development. This protein is one of only a few proteins known in higher vertebrates to contain selenocysteine, which occurs at the active site of glutathione peroxidase and is coded by the nonsense (stop) codon TGA.
• Form: Liquid. In 20mM Tris-HCl buffer (pH 8.0) containing 2mM DTT, 30% glycerol, 100mM NaCl
• Molecular Weight: 24.2kDa (223aa), confirmed by MALDI-TOF
• Purity: > 90% by SDS - PAGE
• Concentration: 0.5mg/ml (determined by Bradford assay)
• Sequences of amino acids: MGSSHHHHHH SSGLVPRGSH MCAARLAAAA AAAQSVYAFS ARPLAGGEPV SLGSLRGKVL LIENVASLCG TTVRDYTQMN ELQRRLGPRG LVVLGFPCNQ FGHQENAKNE EILNSLKYVR PGGGFEPNFM LFEKCEVNGA GAHPLFAFLR EALPAPSDDA TALMTDPKLI TWSPVCRNDV AWNFEKFLVG PDGVPLRRYS RRFQTIDIEP DIEALLSQGP SCA
• Storage: Can be stored at +4C short term (1-2 weeks). For long term storage, aliquot and store at -20C or -70C. Avoid repeated freezing and thawing cycles.
• Pathways: Amyotrophic lateral sclerosis (ALS); Arachidonic acid metabolism; Direct p53 effectors; Folate Metabolism; Glutathione metabolism; Huntington"s disease; Metabolism of nucleotides; Oxidative Stress; Purine catabolism; Selenium Metabolism and Selenoproteins; Selenium Pathway; glutathione redox reactions I
• Publications:
  Impact of carbonylation on glutathione peroxidase-1 activity in human hyperglycemic endothelial cells (2018)

Gene Information

• Gene Name: GPX1 glutathione peroxidase 1 [ Homo sapiens ]
• Official Symbol: GPX1
• Synonyms: GPX1; glutathione peroxidase 1; GSHPX1; MGC14399; MGC88245; GPX1; Cellular glutathione peroxidase; MGC14399; GPx-1; MGC88245; GSHPx-1; OTTHUMP00000210766; OTTHUMP00000210765; EC 1.11.1.9; EC 1.11.1
• Gene ID: 2876
• mRNA Refseq: NM_000581
• Protein Refseq: NP_000572
• MIM: 138320
• UniProt ID: P07203
• Chromosome Location: 3p21.3
• Function: SH3 domain binding; endopeptidase inhibitor activity; glutathione peroxidase activity; oxidoreductase activity

Citation

Impact of carbonylation on glutathione peroxidase-1 activity in human hyperglycemic endothelial cells

Journal: Redox Biology    PubMed ID: 29499564    Data: 2018/3/1

Authors: Cheryl S. Sultan, Andrea Saackel, Andreas H. Wagner

Article Snippet:For some approaches in vitro metal catalyzed oxidation of recombinant GPx1 protein was performed as previously published elsewhere followed by activity measurements or mass spectrometry analysis.For some approaches in vitro metal catalyzed oxidation of recombinant GPx1 protein was performed as previously published elsewhere followed by activity measurements or mass spectrometry analysis.. For this purpose recombinant GPx1 (Creative BioMart, #GPX1-1273H) was dissolved at 0.5 mg/ml in sample buffer (50 mM Tris-HCl, pH 7.6, containing 5 mM EDTA).. Oxidation was accomplished by supplementing 10–20 μl of protein solution (5 μg) with a freshly prepared FeSO4 with a final concentration of 1.2 mmol/L and incubating for 10–30 min at room temperature after addition of 0.05% (w/w) cumene hydroperoxide (Sigma-Aldrich, #247502) or 0.3% (w/w) hydrogen peroxide (Merck Millipore, #107209).Oxidation was accomplished by supplementing 10–20 μl of protein solution (5 μg) with a freshly prepared FeSO4 with a final concentration of 1.2 mmol/L and incubating for 10–30 min at room temperature after addition of 0.05% (w/w) cumene hydroperoxide (Sigma-Aldrich, #247502) or 0.3% (w/w) hydrogen peroxide (Merck Millipore, #107209).

Primers used for qPCR.

Enhanced GPx activity compensates for the decreased GPx1 expression in HUVECs treated with high glucose or MG. HUVECs were exposed to D -glucose (Glc, n = 5) or MG ( n = 4) for 24 h. D -Mannitol ( D -Man) was used as osmotic control for 22 mM D -glucose, MG was scavenged by aminoguanidine (AG, 500 μM). GPx1 protein levels were determined by ( A ) Western blot analysis (the insert shows exemplary Western blots). ( B ) GPx enzymatic activity was measured as indicated in the Material and Methods section, and normalized to the GPx1 protein content. ( C ) For inhibition of the proteasome (exemplary Western blot analysis) cells were pre-incubated for 1 h with 5 nmol/L bortezomib (0.01% in DMSO); therefore a DMSO control was added. *p < 0.05 vs control. Note that the endothelial cell basal medium used as control (contr) itself contained 5.55 mmol/L D -glucose.

Carbonylation-associated increase in GPx activity. ( A ) Specific quantification of GPx1 carbonylation in HUVECs incubated with 22 mmol/L D -glucose (Glc) for 24 h using a sandwich GPx1 ELISA and DNPH-derivatized cell lysates. IgG-antibody (IgG) and a derivatization control solution (contr) were used to demonstrate overall specificity. n = 3, *p < 0.05 vs IgG control. ( B ) Measuring enzymatic activity of a recombinant GPx1 protein (contr) under mild oxidative conditions in the presence of iron (Fe(II)) and MG. Statistical summary, n = 7–13, *p < 0.05 vs control. Fe (II) metal specificity of hydroperoxide-dependent GPx1 carbonylation was demonstrated using comparable concentrations of Fe(III) and Cu(II) ( n = 3). The insert shows recombinant GPx1 in vitro carbonylated under mild (0.05% (w/w) cumene hydroperoxide) or strong (0.3% (w/w) hydrogen peroxide) oxidative conditions and detected by Western blot analysis (exemplary images). Note the aggregated GPx1 protein marked by arrows, which might reflect an oxidative linkage between GPx1 monomers. ( C, D ) Location of the mutated residues in the 3D structure of human GPx1. ( C ) Side view of GPx1 homodimers showing selenic acid at the selenocysteine site (yellow, van der Waals spheres representation), the four EKCE amino acids (stick representation), and the intermolecular distances between the C-alpha atoms (dashed lines). The surface representation of the left monomer in the dimeric structure (white) shows the active site located in a shallow depression around the selenic acid. ( D ) GPx1 dimer interface. The right monomer (gray, cartoon representation) of the dimer structure shown in (C) was rotated by 90° to show the dimer interface. Additionally, the selenic acid is shown in yellow spheres on the monomer along with the intramolecular distances between the corresponding C-alpha atoms for the mutated residues. ( E ) Effects of different mutations on GPx1 enzymatic activity. GPx activity assay with MCF-7 lysates and adenovirally mediated overexpression of wild-type and mutant GPx1 proteins (Ad.GPx1, wild-type protein; Ad.Mut, EKCE replaced by SAIS; Ad.K114, Ad.E116). Ad.eGFP reporter gene expression and NTC (non-transduced control) were used as controls (statistical summary, n = 4, *p < 0.05 vs NTC). The insert shows an exemplary Western blot of recombinant wild-type and mutant GPx1 proteins, demonstrating the presence of roughly equal quantities of overexpressed wild-type and mutant proteins in MCF-7 cells. β-Actin served as a loading control.