Subunits of human .alpha.2-macroglobulin produced by specific reduction of interchain disulfide bonds with thioredoxin

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Abstract
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 .apprx. 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 specially cleaved by nonstoichiometric amounts of trypsin, the isolated subunits had minimal proteinase binding ability. Reaction of subunits with methylamine or typsin produced changes of far-ultraviolet circular dichroism and near-ultraviolet absorption similar to those induced in the whole .alpha.2M molecule, although in contrast with whole .alpha.M 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. The subunits were eliminated from the circulation in the rat only slightly faster than intact .alpha.2M. Reaction of the subunits with methylamine or trypsin increased the clearance rate somewhat, but this rate was still about 10-fold lower than that of trypsin-treated whole .alpha.2M. These observations indicate that specific reduction of the interchain disulfide bonds of .alpha.2M by thioredoxin lead to dissociation of the inhibitor into individual subunits, which retain many properties of intact .alpha.2M but lack proteinase binding ability and the ability to fully expose the receptor recognition site on reaction with amines or proteinases.