Title |
Purification from Placenta, Amino Acid Sequence, Structure Comparisons and cDNA Cloning of Human Glutaredoxin
|
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Published in |
FEBS Journal, March 2005
|
DOI | 10.1111/j.1432-1033.1995.tb20356.x |
Pubmed ID | |
Authors |
C. Alicia Padilla, Emilia Martínez‐Galisteo, J. Antonio Bárcena, Giannis Spyrou, Arne Holmgren |
Abstract |
Glutaredoxin is generally a glutathione-dependent hydrogen donor for ribonucleotide reductase and also catalyses general glutathione (GSH)-disulfide-oxidoreduction reactions in the presence of NADPH and glutathione reductase. A Glutaredoxin from human placenta was purified to homogeneity, as judged by SDS/PAGE and IEF (12 kDa). Purification was monitored by the activity with hydroxyethyl disulfide as a substrate. Values of pI for glutaredoxin were obtained by IEF; the pI of the protein shifted from 7.3 in its fully reduced state to 9.0 in the oxidized state after treatment with excess hydroxyethyl disulfide. The glutaredoxin preparation showed GSH-dependent hydrogen-donor activity with recombinant mouse ribonucleotide reductase, it exhibited dehydroascorbate reductase activity as well as hydroxyethyl-disulfide-reducing activity. The amino acid sequence (residues 3-104) of glutaredoxin was determined by peptide sequencing and residues 1, 2 and 105 by cDNA sequence analysis. The glutaredoxin sequence comprised the classical active site for glutaredoxins -Cys22-Pro-Tyr-Cys25- and three additional half-cystine residues; two of these in positions 78 and 82. The sequence was similar to other known mammalian glutaredoxins (about 80% identities), with important differences such as one additional Cys residue (Cys7) and no Met residue. The sequence of human glutaredoxin was compared to that of Escherichia coli glutaredoxin with known three-dimensional structure in solution to identify conserved residues and predict a structure from alignment. In particular the GSH-binding site of glutaredoxin was conserved between all molecules. A cDNA that encodes the entire glutaredoxin gene (grx) and flanking sequences was isolated from a human spleen cDNA library. The nucleotide sequence of this cDNA (0.8 kb) was determined, including the complete grx gene. |
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Demographic breakdown
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Student > Bachelor | 2 | 8% |
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