Glutathione-dependent synthesis of deoxyribonucleotides. Purification and characterization of glutaredoxin from Escherichia coli.

Abstract

The protein glutaredoxin, which is required for glutathione (GSH)-dependent formation of deoxyribonucleotides by ribonucleotide reductase, has been purified from Escherichia coli B wild type cells. During purification, glutaredoxin behaved as a small, heat-stable, acidic protein which separated from thioredoxin during DEAE-cellulose chromatography. The isolation procedure involved four chromatography steps on DEAEcellulose, Sephadex G-60, and CM-cellulose, and resulted in a 40,000-fold purification of glutaredoxin. The final preparation was homogeneous as judged by polyacrylamide gel electrophoresis and the presence of a single NH,-terminal amino acid. Glutaredoxin has a molecular weight of 11,600 and contains around 89 amino acid residues, including 2 half-cystine residues forming a single disulfide bridge. This cystine disulfide bridge was readily reduced to a dithiol by GSH, NADPH, and glutathione reductase but was not reduced with NADPH and thioredoxin reductase from E. coli. Tryptic peptide maps of chemically reduced and carboxymethylated glutaredoxin and E. coli thioredoxin demonstrated that the two proteins are structurally unrelated, and thus are the products of two separate genes. Glutaredoxin has two enzymatic activities dependent on GSH. One couples oxidation of GSH to reduction of ribonucleotides by ribonucleotide reductase; the other is a powerful GSH-disulfide transhydrogenase. Crude extracts of E. coli contain a family of unknown enzymes with GSH-transhydrogenase activity and glutaredoxin represents only a small fraction of the total activity of these.