Purification from Placenta, Amino Acid Sequence, Structure Comparisons and cDNA Cloning of Human Glutaredoxin

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 indentify 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. Copyright © 1995, Wiley Blackwell. All rights reserved