Pizzo S V, Roche P A, Feldman S R, Gonias S L
Biochem J. 1986 Aug 15;238(1):217-25. doi: 10.1042/bj2380217.
alpha 2-Macroglobulin (alpha 2M)-methylamine that had been allowed to react with cis-dichlorodiammineplatinum(II) (cis-DDP) bound with greatly reduced affinity to specific alpha 2M 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. alpha 2M-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. alpha 2M-methylamine was affected significantly less by the H2O2 exposure after pretreatment with cis-DDP. Amino acid analysis indicated that H2O2 treatment of alpha 2M modified 19 of the 25 methionine residues per alpha 2M subunit. Pretreatment with cis-DDP protected two to four of these methionine residues. The only other residue altered by H2O2 treatment of alpha 2M 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 alpha 2M. Fast-form alpha 2M was still recognized by the alpha 2M 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 alpha 2M with hydroxylamine reversed derivatization of 43 of the 53 histidine residues. Moreover, this treatment also resulted in an alpha 2M fast-form preparation that was recognized only by the alpha 2M receptor. It is concluded that cis-DDP and H2O2 modify a critical methionine residue in the primary sequence of the alpha 2M-receptor recognition site.
α2-巨球蛋白（α2M）-甲胺与顺式二氯二氨铂（II）（顺铂）反应后，其与特定α2M受体的结合亲和力大幅降低，这在体外巨噬细胞结合研究和体内血浆清除实验中得到了证实。随后与二乙基二硫代氨基甲酸盐反应可完全恢复受体识别功能。当二乙基二硫代氨基甲酸盐浓度为总铂浓度的两倍时，可获得最佳效果。经体内和体外研究表明，与过氧化氢反应的α2M-甲胺对特定细胞表面结合位点的竞争效果较差。暴露于过氧化氢15分钟后，表观解离常数增加了近7倍。顺铂预处理后，α2M-甲胺受过氧化氢暴露的影响明显较小。氨基酸分析表明，过氧化氢处理α2M会使每个α2M亚基的25个甲硫氨酸残基中的19个发生修饰。顺铂预处理可保护其中2至4个甲硫氨酸残基。过氧化氢处理α2M后，唯一发生改变的其他残基是组氨酸。观察到每个亚基的组氨酸残基净减少两个，但顺铂预处理并未改变这一结果。为了排除组氨酸修饰可能导致观察到的过氧化氢诱导的受体识别丧失的微小可能性，使用焦碳酸二乙酯作为组氨酸修饰试剂。该处理修饰了天然和快速形式的α2M中的53个组氨酸残基。经体内和体外研究确定，快速形式的α2M仍可被α2M受体识别；然而，现在一部分修饰后的蛋白质也通过酰基低密度脂蛋白受体清除。焦碳酸二乙酯处理的α2M与羟胺反应可使53个组氨酸残基中的43个去衍生化。此外，该处理还产生了仅被α2M受体识别的α2M快速形式制剂。得出的结论是，顺铂和过氧化氢修饰了α2M受体识别位点一级序列中的关键甲硫氨酸残基。
