PURIFICATION FROM PLACENTA, AMINO-ACID-SEQUENCE, STRUCTURE COMPARISONS AND CDNA CLONING OF HUMAN GLUTAREDOXIN

Glutaredoxin is generally a glutathione-dependent hydrogen donor for r ibonucleotide reductase and also catalyses general glutathione (GSH)-d isulfide-oxidoreduction reactions in the presence of NADPH and glutath ione reductase. A Glutaredoxin from human placenta was purified to hom ogeneity, as judged by SDS/PAGE and IEF (12 kDa). Purification was mon itored by the activity with hydroxyethyl disulfide as a substrate. Val ues 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 st ate after treatment with excess hydroxyethyl disulfide. The glutaredox in preparation showed GSH-dependent hydrogen-donor activity with recom binant mouse ribonucleotide reductase, it exhibited dehydroascorbate r eductase activity as well as hydroxyethyl-disulfide-reducing activity. The amino acid sequence (residues 3-104) of glutaredoxin was determin ed by peptide sequencing and residues 1, 2 and 105 by cDNA sequence an alysis. The glutaredoxin sequence comprised the classical active site for glutaredoxins -Cys22-Pro-Tyr-Cys25- and three additional half-cyst ine residues; two of these in positions 78 and 82. The sequence was si milar to other known mammalian glutaredoxins (about 80% identities), w ith important differences such as one additional Cys residue (Cys7) an d no Met residue. The sequence of human glutaredoxin was compared to t hat of Escherichia coli glutaredoxin with known three-dimensional stru cture in solution to indentify conserved residues and predict a struct ure from alignment. In particular the GSH-binding site of glutaredoxin was conserved between all molecules. A cDNA that encodes the entire g lutaredoxin gene (grx) and flanking sequences was isolated from a huma n spleen cDNA library. The nucleotide sequence of this cDNA (0.8 kb) w as determined, including the complete grx gene.