Abstract
Glutathione-dependent synthesis of deoxyribonucleotides. Purification and characterization of glutaredoxin from Escherichia coli.
Highlights
The first steps in the present method were designed to purify both thioredoxin and glutaredoxin
Peptide Map-Reduction of glutaredoxin by excess dithiothreitol followed by carboxymethylation with [14C]iodoacetic acid resulted in incorporation of 176 nmol of carboxymethylcysteine/mg of protein
Presence of a Cystine Disulfide Bridge in Glutaredoxin-The amino acid analysis and carboxymethylation experiments suggested that glutaredoxin contains a functional disulfide bridge made up from 2 half-cystine residues in the protein (Fig. 11)
Summary
Purification of glutaredoxin from a crude lysate of E. coli tsnC 7004 cells through some batch steps was reported previously [1]. Starting from 0.5 to 1 kg of E. coli tsnC 7004 cells, development of further purification steps, to be described below, resulted in small quantities of a highly purified material in low yield. This material was estimated from polyacrylamide gel electrophoresis to be about 60% pure. The initial large scale batch steps including the ammonium sulfate precipitation were performed at the Microbiological Research Establishment, Porton, Salisbury, Wiltshire, England. Centrifugations in these steps were performed with a Sharples No 26 continuous flow, water-cooled centrifuge. After an additional stirring period of 15 min, the precipitate (6.5 kg) was removed by centrifugation. fraction (Fraction II) was saved
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