Ca. Padilla et al., PURIFICATION FROM PLACENTA, AMINO-ACID-SEQUENCE, STRUCTURE COMPARISONS AND CDNA CLONING OF HUMAN GLUTAREDOXIN, European journal of biochemistry, 227(1-2), 1995, pp. 27-34
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.