Cytotechnology. 1997 Nov;25(1-3):173-82. doi: 10.1023/A:1007920018109.
To explore potential molecular chaperones involved in the intracellular assembly of laminin chains, bovine aortic endothelial cells were treated with a thiol cleavable divalent cross-linking reagent, dithio-bis-(succinimidylpropionate), and cellular proteins cross-linked to laminin chains were co-immunoprecipitated with anti-laminin antiserum. Sodium dodecylsulfate (SDS) gel electrophoresis of the precipitate under reducing condition showed polypeptides with estimated sizes of 80, 60 and 50 kDa together with laminin chains. Two dimensional electrophoresis, in which non-reducing and reducing SDS electrophoresis were combined, suggested that many molecules of these polypeptides were cross-linked to each laminin chain. Sepharose CL-4B beads conjugated with E8 fragment of mouse laminin-1 was prepared. Affinity chromatography with the beads of microsomal proteins from rat liver showed that Bip and HSP70 associated to laminin chains and dissociated upon ATP hydrolysis. Protein-disulfide isomerase also showed affinity to the column. GRP94 and calnexin showed strong affinity and were washed out only with a detergent solution. Thus, many molecular chaperones are suggested to be involved in the intracellular assembly of laminin chains.
为了探索参与层粘连蛋白链细胞内组装的潜在分子伴侣，用硫醇裂解的二价交联试剂二硫代双（琥珀酰亚胺基丙酸酯）处理牛主动脉内皮细胞，并与抗层粘连蛋白抗血清共免疫沉淀与层粘连蛋白链交联的细胞蛋白。在还原条件下沉淀的 SDS 凝胶电泳显示出估计大小为 80、60 和 50 kDa 的多肽与层粘连蛋白链一起。二维电泳，其中将非还原和还原 SDS 电泳结合在一起，表明这些多肽的许多分子与每个层粘连蛋白链交联。用小鼠层粘连蛋白-1 的 E8 片段制备了琼脂糖 CL-4B 珠。用珠亲和层析从小鼠肝微粒体蛋白显示 Bip 和 HSP70 与层粘连蛋白链结合，并在 ATP 水解时解离。蛋白二硫键异构酶也显示出对该柱的亲和力。GRP94 和 calnexin 表现出强烈的亲和力，只有用洗涤剂溶液才能洗脱。因此，许多分子伴侣被认为参与了层粘连蛋白链的细胞内组装。
