The in vivo distribution of oxidized and reduced thioredoxin in Escherichia coli

Abstract

The oxidation state of thioredoxin (oxidized or reduced, thioredoxin-S2 or thioredoxin-(SH)2) in Escherichia coli cells including the degree of thiolphosphorylation has been studied. Previously published experiments (Conley, R. R., and Pigiet, V. (1978) J. Biol. Chem. 253, 5568-5572) suggested that nearly all (96%) thioredoxin molecules in vivo were thiol phosphorylated in growing cells. We have not been able to isolate any phosphothioredoxin using a variety of experimental conditions. Growth of E. coli cells in the presence of 32P-labeled inorganic phosphate followed by purification of thioredoxin by immunoadsorbent chromatography resulted in co-elution with variable amounts of phosphate. The phosphate was not covalently linked to thioredoxin which was isolated in its oxidized disulfide form. Methods to determine the oxidation state of thioredoxin in extracts and cells were developed. These are based on alkylation of thioredoxin-(SH)2 with excess iodoacetic acid followed by purification of total thioredoxin by immunoadsorbent chromatography. The total quantity of thioredoxin was determined by rocket immunoelectrophoresis and the amount of thioredoxin-S2 was measured by its enzymatic activity with thioredoxin reductase. Native polyacrylamide gel electrophoresis was used to separate thioredoxin-S2 and monocarboxymethylated thioredoxin derived from thioredoxin-(SH)2. In crude cell-free extracts, most of the thioredoxin (60-90%) is oxidized. In freshly harvested cells that were permeabilized and treated with [14C] iodoacetate, lower values for thioredoxin-S2 (30-40%) were obtained. No phosphothioredoxin (less than 3%) was detectable.