title: Redox states of DsbA in the periplasm of Escherichia coli.
abstract: DsbA is a periplasmic, disulfide bond formation factor of E. coli. We studied in vivo redox states of its active site cysteines. When periplasmic contents were prepared from iodoacetic acid-treated cells, according to the previously published procedures, variable but major proportions of DsbA were in the reduced form. We found that this was due to an artificial reduction that occurred after cell disruption; even purified and oxidized DsbA underwent reduction when incubated with cell extracts in the absence of any added reducing agent. Such DsbA-reducing activities were detected in both the periplasmic and the cytoplasmic fractions. To circumvent the artifact, we analyzed redox states of DsbA under denaturing conditions. Now virtually all the DsbA molecules were detected as oxidized or reduced in the dsbB+ background or in the dsbB- background, respectively. Using the improved method, we also examined redox states of DsbA when it was overproduced, and followed the oxidation/reduction pathway that DsbA follows after biosynthesis. It is suggested that newly synthesized DsbA is rapidly oxidized by pre-existing DsbA, while oxidation of mature (functional) DsbA requires DsbB, whose roles might include that of antagonizing the actions of DsbA-reducing enzyme(s).
authors.affiliation: Department of Cell Biology, Kyoto University, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Kishigami
authors.forename: Y
authors.initials: Y
authors.lastname: Akiyama
authors.forename: K
authors.initials: K
authors.lastname: Ito
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D007535:Isomerases / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D011994:Recombinant Proteins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases