Isolation and characterization of phosphothioredoxin from Excherichia coli.

Thioredoxin was isolated as a phosphoprotein from actively growing cultures of Escherichia coli. Labeling was performed in vivo by growing cells in the presence of 32P-labeled inorganic phosphate, and phosphothioredoxin was purified in one step by immunoabsorption to a thioredoxin antibody column. The stoichiometry of phosphate bound was 0.7 to 0.8 mol of phosphate/mol of thioredoxin. The phospho-amino acid linkage was identified as a thiol phosphate by several criteria: (a) the maximum lability of the phosphate bond was between pH 2.5 and 3.5 (t1/2 (37 degrees) = 200 h (pH 7 to 8); 0.4 h (pH 3.0); 200 h (pH 1.0)); (b) the phosphate linkage was very labile in the presence of iodine at neutral pH (t1/2 less than 1 min); and the phosphopeptide was identified as Cys32-Gly-Pro-Cys35-Lys, the same sequence previously implicated as the active site for disulfide-linked oxidation-reduction reactions. Phosphate was distributed on either cysteine, with 60% of the phosphate bound to cysteine32. Results are discussed in terms of the possible role of phosphothioredoxin as an intermediate in phosphotransferase reactions.