Reconstitution of Escherichia coli thioredoxin from complementing peptide fragments obtained by cleavage at methionine-37 or arginine-73.

摘要: Abstract Thioredoxin from Escherichia coli (a small hydrogen transport protein containing 108 amino acid residues and having in its oxidized form a single disulfide bond) was acylated with citraconic anhydride. Citraconylation of all groups resulted total loss enzymatic activity thioredoxin reductase immunoprecipitin antithioredoxin antibodies; both these activities were fully restored after deblocking the citraconylated by treatment. Large enzymatically inactive peptide fragments prepared selective cleavage at Arg-73 Met-37, respectively, tested for their antigenic antibodies against native thioredoxin. Thioredoxin-T-(1-73) thioredoxin-T-(74-108) separated Sephadex G-50 chromatography 50% acetic deblocked trypsin digest afforded about 25% corresponding immunoprecipitate without significant inhibition antigen excess. Thioredoxin-T-(74-108) gave no but potent inhibitor reaction as measured turbidity formation. A major determinant present COOH-terminal sequence molecule. An equimolar mixture thioredoxin-T-(1-73) showed full immunoprecipitation reductase. Gel experiments pH 8 main symmetrical peak elution volume composition identical The results strongly suggested reconstitution two to complex, thioredoxin-T', conformation similar Reconstitution thioredoxin-like structure also obtained overlapping thioredoxin-C-(38-108), although peptides represented more than protein. Previous thioredoxin-C-(1-37) thioredoxin-C-(38-108) complex called thioredoxin-C' (Holmgren, A. (1972) Fed. Eur. Biochem. Soc. Lett. 24, 351-354). Together results, this shows that three different combinations larger permissible sites gives In each case, least one component strong immunochemical thioredoxin, Netion tetrameric dimeri