Further characterization of the platinum-reactive component of the alpha 2-macroglobulin-receptor recognition site.
Further characterization of the platinum-reactive component of the alpha 2-macroglobulin-receptor recognition site.
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α2-巨球蛋白受体识别位点的铂反应成分的进一步表征。
DOI：
10.1042/bj2380217
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Gonias,SL
中科院分区：
文献类型：
--
作者：
Pizzo,SV;Roche,PA;Feldman,SR;Gonias,SL
通过体外巨噬细胞结合研究和体内血浆清除实验确定，α 2-巨球蛋白（α 2 M）-甲胺与顺式-二氯二氨铂（II）（顺式-DDP）反应后，与特异性α 2 M受体的亲和力大大降低。随后与二乙基二硫代氨基甲酸酯的反应完全恢复了受体识别功能。当二乙基二硫代氨基甲酸盐浓度为总铂浓度的两倍时，获得最佳效果。体内和体外研究均表明，允许与H2 O2反应的α 2 M-甲胺对特定细胞表面结合位点的竞争效率较低。的表观解离常数增加了近7倍，通过15分钟的暴露于H2 O2。用顺式DDP预处理后，α 2 M-甲胺受H2 O2暴露的影响显著较小。氨基酸分析表明，过氧化氢处理α 2 M修改19的25甲硫氨酸残基每个α 2 M亚基。用顺式DDP预处理保护了这些甲硫氨酸残基中的两到四个。通过α 2 M的H2 O2处理改变的唯一其他残基是组氨酸。观察到每个亚基两个组氨酸残基的净减少，但顺式DDP预处理没有改变这一结果。为了排除组氨酸修饰可能导致观察到的H2 O2诱导的受体识别损失的微小可能性，采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α 2 M中的53个组氨酸残基。通过体内和体外研究确定，快速型α 2 M仍然被α 2 M受体识别;然而，一部分修饰蛋白现在也通过酰基低密度脂蛋白受体清除。经焦碳酸二乙酯处理的α 2 M与羟胺反应，对53个组氨酸残基中的43个进行反向衍生。此外，该处理还产生了仅被α 2 M受体识别的α 2 M快型制剂。它的结论是，顺式DDP和H2 O2修改一个关键的甲硫氨酸残基的α 2 M-受体识别位点的一级序列。
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.