Further characterization of the platinum-reactive component of the α2-macroglobulin-receptor recognition site

Abstract

α2-Macroglobulin (α2M)-methylamine that had been allowed to react with cis-dichlorodiammineplatinum(II) (cis-DDP) bound with greatly reduced affinity to specific α2-M receptors, as determined by macrophage binding studies in vitro and plasma-clearance experiments in vivo. Subsequent reaction with diethyl dithiocarbamate completely restored receptor recognition function. The optimal effect was obtained when the diethyl dithiocarbamate concentration was twice the total platinum concentration. α2M-methylamine that was allowed to react with H2O2 competed less effectively for specific cell-surface binding sites, as demonstrated by studies both in vivo and in vitro. The apparent dissociation constant was increased nearly 7-fold by a 15 min exposure to H2O2. α2M-methylamine was affected significantly less by the H2O2 exposure after pretreatment with cis-DDP. Amino acid analysis indicated that H2O2 treatment of α2M modified 19 of the 25 methionine residues per α2M subunit. Pretreatment with cis-DDP protected two to four of these methionine residues. The only other residue altered by H2O2 treatment of α2M was histidine. A net decrease of two histidine residues per subunit was observed, but cis-DDP pretreatment did not alter this result. In order to rule out the slight possibility that histidine modification might account for the observed H2O2-induced loss in receptor recognition, diethyl pyrocarbonate was employed as a histidine-modifying reagent. This treatment modified 53 histidine residues in both native and fast-form α2M. Fast-form α2M was still recognized by the α2M receptor, as determined by studies both in vivo and in vitro; however, a fraction of the modified protein now cleared via the acyl-low-density-lipoprotein receptor as well. Reaction of diethyl pyrocarbonate-treated α2M with hydroxylamine reversed derivatization of 43 of the 53 histidine residues. Moreover, this treatment also resulted in an α2M fast-form preparation that was recognized only by the α2M receptor. It is concluded that cis-DDP and H2O2 modify a critical methionine residue in the primary sequence of the α2M-receptor recognition site.