Intersubunit cross-linking by cis-dichlorodiammineplatinum(II) stabilizes an alpha 2-macroglobulin "nascent" state: evidence that thiol ester bond cleavage correlates with receptor recognition site exposure.
Intersubunit cross-linking by cis-dichlorodiammineplatinum(II) stabilizes an alpha 2-macroglobulin "nascent" state: evidence that thiol ester bond cleavage correlates with receptor recognition site exposure.
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DOI：
10.1021/bi00402a040
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发表时间：
1988-01
期刊：
影响因子：
2.9
通讯作者：
P. Roche;P. E. Jensen;S. Pizzo
中科院分区：
文献类型：
--
作者：
P. Roche;P. E. Jensen;S. Pizzo
用蛋白酶处理人α 2-巨球蛋白（α 2M）导致α 2M亚基的分裂，随后导致抑制剂的构象改变。这种变化不可逆地捕获了蛋白酶，并伴随着四个巯基的产生以及受体识别位点的暴露。顺式二氯二胺铂（II）（顺式ddp）引起广泛的亚基间交联α 2M。α - 2M或顺式ddp处理的α - 2M与胰蛋白酶孵育可导致完全的亚基切割；然而，通过非变性聚丙烯酰胺凝胶电泳（PAGE）、受体识别位点暴露或抑制剂硫基的出现，胰蛋白酶处理顺式ddp -alpha 2M不会导致构象改变。这些结果与先前的研究结果形成鲜明对比，这些研究表明，顺式ddp处理的alpha 2M与CH3NH2孵育导致巯基酯键断裂和受体识别位点暴露。顺式ddp处理的α 2M仅结合0.13 mol的125i -胰蛋白酶/mol顺式ddp - α 2M。胰蛋白酶处理的顺式- ddp - α 2M与二乙基二硫代氨基甲酸酯（DDC）（一种铂化合物的强螯合剂）孵育，可以去除亚基间交联并完成α 2M构象变化，这是由非变性PAGE确定的。完全的受体识别位点暴露和3.3巯基/mol α 2M的出现也发生在这种处理之后。这些结果表明，顺式ddp交联α - 2M可以阻止抑制剂的构象变化，这是硫醇酯键激活和裂解所必需的。（摘要删节250字）
Treatment of human alpha 2-macroglobulin (alpha 2M) with proteinase results in cleavage of the alpha 2M subunits and subsequently in a conformational change in the inhibitor. This change irreversibly traps the proteinase and is accompanied by the generation of four thiol groups as well as exposure of receptor recognition sites. cis-Dichlorodiammineplatinum(II) (cis-DDP) causes extensive intersubunit cross-linking of alpha 2M. Incubation of alpha 2M or cis-DDP-treated alpha 2M with trypsin results in complete subunit cleavage; however, trypsin treatment of cis-DDP-alpha 2M does not result in a conformational change as determined by nondenaturing polyacrylamide gel electrophoresis (PAGE), receptor recognition site exposure, or appearance of thiol groups from the inhibitor. These results are in marked contrast to previous studies which demonstrated that incubation of cis-DDP-treated alpha 2M with CH3NH2 resulted in thiol ester bond cleavage and receptor recognition site exposure. cis-DDP-treated alpha 2M bound only 0.13 mol of 125I-trypsin/mol of cis-DDP-alpha 2M. Incubation of trypsin-treated cis-DDP-alpha 2M with diethyldithiocarbamate (DDC), a potent chelator of platinum compounds, results in the removal of the intersubunit cross-links and completion of the alpha 2M conformational change as determined by nondenaturing PAGE. Complete receptor recognition site exposure and the appearance of 3.3 thiol groups/mol of alpha 2M also occur following this treatment. These results demonstrate that cross-linking of alpha 2M by cis-DDP prevents a conformational change in the inhibitor which is necessary for thiol ester bond activation and cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)