Subunits of human alpha 2-macroglobulin produced by specific reduction of interchain disulfide bonds with thioredoxin.
Disulfide bonds in alpha 2-macroglobulin (alpha 2M) were reduced with the thioredoxin system from Escherichia coli. Under the conditions selected, 3.5-4.1 disulfide bonds were cleaved in each alpha 2M molecule, as determined by the consumption of NADPH during the reaction and by the incorporation of iodo[3H]acetate into the reaction product. This extent of disulfide bond reduction, approximately corresponding to that expected from specific cleavage of all four interchain disulfide bonds of the protein, coincided with the nearly complete dissociation of the intact alpha 2M molecule to a species migrating as an alpha 2M subunit in gel electrophoresis, under both denaturing and nondenaturing conditions. The dissociation was accompanied by only small changes of the spectroscopic properties of the subunits, which thus retain a near-native conformation. Reaction of isolated subunits with methylamine or trypsin led to the appearance of approximately 0.55 mol of thiol group/mol of subunits, indicating that the thio ester bonds are largely intact. Moreover, the rate of cleavage of these bonds by methylamine was similar to that in the whole alpha 2M molecule. Although the bait region was specifically cleaved by nonstoichiometric amounts of trypsin, the isolated subunits had minimal proteinase binding ability. Reaction of subunits with methylamine or trypsin produced changes of farultraviolet circular dichroism and near-ultraviolet absorption similar to those induced in the whole alpha 2M molecule, although in contrast with whole alpha 2M no fluorescence change was observed. The methylamine- or trypsin-treated subunits reassociated to a tetrameric species, migrating as the "fast" form of whole alpha 2M in gradient gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)
D001426:Bacterial Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013879:Thioredoxins / Q000378:metabolism*; D014357:Trypsin; D000511:alpha-Macroglobulins / Q000378:metabolism*
D001426:Bacterial Proteins; D004220:Disulfides; D046911:Macromolecular Substances; D000511:alpha-Macroglobulins; D013879:Thioredoxins; D014357:Trypsin