Formation of sodium dodecyl sulfate-stable fibronectin multimers - Failure to detect products of thiol-disulfide exchange in cyanogen bromide or limited acid digests of stabilized matrix fibronectin
Formation of sodium dodecyl sulfate-stable fibronectin multimers - Failure to detect products of thiol-disulfide exchange in cyanogen bromide or limited acid digests of stabilized matrix fibronectin
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DOI：
10.1074/jbc.271.15.9084
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发表时间：
1996-04-12
影响因子：
4.8
通讯作者：
Mosher, DF
中科院分区：
文献类型：
--
作者：
Chen, F;Mosher, DF
纤连蛋白以可溶形式存在于体液中并作为细胞外基质的纤维状组分。基质纤连蛋白在SDS中以大的复合物缔合，除非还存在还原剂。该观察结果表明复合物的形成是由于通过硫醇-二硫键交换形成的原聚体间二硫化物。为了定位推定的新二硫化物，我们通过氯胺-T方法或用I-125-Bolton-Hunter试剂标记原聚体纤连蛋白，将I-125-纤连蛋白掺入培养的成纤维细胞的基质中，并将基质纤连蛋白进行酸或溴化氰消化，当用Bolton-Hunter试剂标记的基质I-125-纤连蛋白和原聚体I-125-纤连蛋白的溴化氰消化物在SDS中通过二维聚丙烯酰胺凝胶电泳分析时，第一维是非还原性的，第二维是还原性的，我们不能鉴定在第一维中作为高分子量复合物迁移的基质纤连蛋白的任何片段。基质I-125-纤连蛋白的有限酸消化也解离了大多数高分子量复合物。由于我们可以解释纤连蛋白中含有半胱氨酸或胱氨酸的所有部分，我们得出结论，基质纤连蛋白不稳定的interprotomeric二硫化物。相反，我们建议，稳定化是由非共价蛋白质-蛋白质相互作用介导的，这些相互作用对还原、溴化氰消化或有限的酸消化敏感。
Fibronectin exists in a soluble form in body fluids and as a fibrillar component of the extracellular matrix. Matrix fibronectin associates as large complexes in SDS unless a reducing reagent is also present, This observation suggests that complex formation is due to interprotomeric disulfides that form by thiol-disulfide exchange, To localize the presumptive new disulfides, we labeled protomeric fibronectin by the chloramine-T method or with I-125-Bolton-Hunter reagent, incorporated I-125-fibronectin into the matrix of cultured fibroblasts, and subjected matrix fibronectin to acid or cyanogen bromide digestion, When cyanogen bromide digests of matrix I-125-fibronectin and protomeric I-125-fibronectin labeled with Bolton-Hunter reagent were analyzed by two-dimensional polyacrylamide gel electrophoresis in SDS, with the first dimension being nonreducing and the second reducing, we were not able to identify any fragments of matrix fibronectin that migrated as high molecular weight complexes in the first dimension. Limited acid digestion of matrix I-125-fibronectin also dissociated the majority of the high molecular weight complexes. Since we could account for all of the parts of fibronectin that contain cysteine or cystine, we conclude that matrix fibronectin is not stabilized by interprotomeric disulfides. We propose, instead, that stabilization is mediated by noncovalent protein-protein interactions that are sensitive to reduction, cyanogen bromide digestion, or limited acid digestion.