Malinoff H L, Wicha M S
J Cell Biol. 1983 May;96(5):1475-9. doi: 10.1083/jcb.96.5.1475.
We used affinity chromatography to isolate a specific laminin-binding protein from murine fibrosarcoma cells. These cells bind exogenous laminin to their surface with high affinity (Kd = 2 X 10(-9)M for laminin) with approximately 5 X 10(4) sites per cell. Laminin affinity chromatography of [35S]methionine-labeled cell extracts produced two distinct proteins. One was identified as Type IV (basement membrane) collagen based on its migration pattern on SDS gels and bacterial collagenase sensitivity. The other protein, which migrates as a single band or closely spaced doublet on reduced SDS gels, has a reduced molecular weight of 69,000. Using a nitrocellulose filter disk assay, we found that the latter protein specifically bound 125I-laminin with the same high affinity (Kd = 2 X 10(-9)M for laminin) as did intact fibrosarcoma cells. By iodinating intact cells, we demonstrated that this laminin-binding protein is on the cell surface. We conclude that this protein with reduced molecular weight of 69,000 is a subunit or component of a larger cell surface receptor protein for laminin in this fibrosarcoma model. This laminin receptor may mediate the interaction of the cell with its extracellular matrix.
我们利用亲和层析法从鼠纤维肉瘤细胞中分离出一种特异性层粘连蛋白结合蛋白。这些细胞以高亲和力（层粘连蛋白的解离常数Kd = 2×10⁻⁹M）将外源性层粘连蛋白结合到其表面，每个细胞约有5×10⁴个结合位点。对[³⁵S]甲硫氨酸标记的细胞提取物进行层粘连蛋白亲和层析，产生了两种不同的蛋白质。一种根据其在SDS凝胶上的迁移模式和对细菌胶原酶的敏感性被鉴定为IV型（基底膜）胶原。另一种蛋白质在还原SDS凝胶上以单一条带或紧密间隔的双条带形式迁移，分子量降低为69,000。使用硝酸纤维素滤膜圆盘试验，我们发现后一种蛋白质与¹²⁵I - 层粘连蛋白特异性结合，其亲和力与完整的纤维肉瘤细胞相同（层粘连蛋白的Kd = 2×10⁻⁹M）。通过对完整细胞进行碘化，我们证明这种层粘连蛋白结合蛋白位于细胞表面。我们得出结论，这种分子量降低为69,000的蛋白质是该纤维肉瘤模型中层粘连蛋白较大细胞表面受体蛋白的一个亚基或组成部分。这种层粘连蛋白受体可能介导细胞与其细胞外基质的相互作用。
