Vartio T, Barlati S, de Petro G, Miggiano V, Stähli C, Takács B, Vaheri A
Eur J Biochem. 1983 Sep 15;135(2):203-7. doi: 10.1111/j.1432-1033.1983.tb07638.x.
Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin-binding and heparin-binding fractions were analyzed in NaDodSO4-polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti-fibronectin antibody. In early cathepsin G digests, several gelatin-binding fragments were detected: a few large (Mr greater than or equal to 150 000) polypeptides and fragments of Mr = 85 000, 72 000, 64 000 and 40 000. The 85 000-Mr and 64 000-Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72 000-Mr and 40 000-Mr fragments did not. Therefore the 64 000-Mr fragments are likely to be derived from the 85 000-Mr fragments. Three large fragments that bound to heparin, but not to gelatin were detected: Mr = 145 000, 135 000 and 120 000. Of these only the 135 000-Mr peptide reacted with the antibody. When fibronectin was digested with thrombin, polypeptides of Mr = 180 000-200 000 and a 30 000-Mr NH2-terminal fragment were produced. Cathepsin G added to this mixture further cleaved the fragments to a digestion pattern resembling that obtained from intact fibronectin except that the 85 000-Mr and 64 000-Mr fragments appeared as single bands and the amount of the 72 000-Mr fragment was reduced. The results suggest that thrombin cleaves the 30 000-Mr fragment preferentially from the NH2-terminal end of one of the two subunits of fibronectin and that the 85 000-Mr, 72 000-Mr and 64 000-Mr fragments obtained by the additional cathepsin G digestion were derived from the other chain. The results are consistent with the model that the antigenic determinant resides 72 000-85 000 Da from the NH2-terminus and is cleaved by cathepsin G alternatively at one of its sides. Thus, the components of the 85 000-Mr and 64 000-Mr doublets are derived from different subunits and the region located by the antibody may be responsible for the difference in their migration in the polyacrylamide gel.
纯化的血浆纤连蛋白先后用凝血酶和组织蛋白酶G消化，或仅用组织蛋白酶G消化，然后在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中分析降解产物及其明胶结合和肝素结合部分，随后用一种特定的抗纤连蛋白单克隆抗体进行免疫印迹分析。在组织蛋白酶G早期消化产物中，检测到几个明胶结合片段：几个大的（分子量大于或等于150000）多肽以及分子量为85000、72000、64000和40000的片段。分子量为85000和64000的片段呈现为紧密相邻的双峰，并与抗体发生反应，而分子量为72000和40000的片段则不反应。因此，分子量为64000的片段可能源自分子量为85000的片段。检测到三个与肝素结合但不与明胶结合的大片段：分子量分别为145000、135000和120000。其中只有分子量为135000的肽段与抗体发生反应。当纤连蛋白用凝血酶消化时，产生了分子量为180000 - 200000的多肽和一个分子量为30000的氨基末端片段。向该混合物中加入组织蛋白酶G进一步将片段切割成类似于从完整纤连蛋白获得的消化模式，只是分子量为85000和64000的片段呈现为单条带，且分子量为72000的片段量减少。结果表明，凝血酶优先从纤连蛋白两个亚基之一的氨基末端切割分子量为30000的片段，并且通过额外的组织蛋白酶G消化获得的分子量为85000、72000和64000的片段源自另一条链。这些结果与以下模型一致：抗原决定簇位于距氨基末端72000 - 85000道尔顿处，并被组织蛋白酶G在其一侧交替切割。因此，分子量为85000和64000双峰的组分源自不同亚基，并且抗体定位的区域可能是它们在聚丙烯酰胺凝胶中迁移差异的原因。
