Isemura M, Yosizawa Z, Takahashi K, Kosaka H, Kojima N, Ono T
J Biochem. 1981 Jul;90(1):1-9. doi: 10.1093/oxfordjournals.jbchem.a133437.
Fibronectin was isolated from porcine plasma by affinity chromatography with gelatin-linked Sepharose 4B. Porcine fibronectin had a chemical composition similar to those of human and other fibronectins and reacted with antiserum raised against human fibronectin. It showed hemagglutination activity with trypsin-treated rabbit erythrocytes, though the activity was far less than that of human fibronectin. Porcine plasma fibronectin consisted of two subunit chains of about 230,000-daltons linked by disulfide bonds(s). Limited proteolysis of this protein with porcine liver cathepsin B yielded five major fragments which were investigated by affinity chromatography with gelatin- and heparin-linked Sepharose 4B. One fragment (Mr = 50,000) was bound to gelatin but not to heparin, while the remaining four were bound to heparin but not to gelatin, suggesting that plasma fibronectin takes a discrete domain structure with respect to interaction with these two macromolecules. The three larger heparin-binding fragments, Mr = 175,000, 150,000, and 130,000 were eluted with different concentrations of a mixture of NaCl and urea from the heparin-column, suggesting that they have different interactions with heparin, the 130,000-dalton fragment being the one with the strongest interaction. After reduction with 2-mercaptoethanol, the 175,000-dalton fragment was converted to the 150,000-dalton region fragment, which, together with the unchanged 150,000-dalton fragment, appeared to be equivalent in amount to the 130,000-dalton fragment. This finding suggests that the 150,000- and 130,000-dalton fragments may have originated from different subunit chains. Since the 175,000-dalton fragment was not produced by cathepsin B digestion of fibronectin which had been treated with plasmin, it was concluded that the 175,000-dalton fragment contained interchain disulfide bond(s) which had linked the native subunit chains. These results suggest that porcine plasma fibronectin has non-identical subunit chains composed of domains which differ in interaction with heparin and in susceptibility to cathepsin B.
通过与明胶偶联的琼脂糖凝胶4B进行亲和层析，从猪血浆中分离出纤连蛋白。猪纤连蛋白的化学组成与人和其他纤连蛋白相似，并能与抗人纤连蛋白的抗血清发生反应。它对经胰蛋白酶处理的兔红细胞表现出血凝活性，不过该活性远低于人纤连蛋白。猪血浆纤连蛋白由两条约230,000道尔顿的亚基链通过二硫键连接而成。用猪肝组织蛋白酶B对该蛋白进行有限的蛋白水解，产生了五个主要片段，通过与明胶和肝素偶联的琼脂糖凝胶4B进行亲和层析对其进行了研究。一个片段（Mr = 50,000）与明胶结合但不与肝素结合，而其余四个片段与肝素结合但不与明胶结合，这表明血浆纤连蛋白在与这两种大分子相互作用方面具有离散的结构域结构。三个较大的肝素结合片段，Mr = 175,000、150,000和130,000，从肝素柱上用不同浓度的NaCl和尿素混合物洗脱，这表明它们与肝素的相互作用不同，130,000道尔顿的片段与肝素的相互作用最强。用2-巯基乙醇还原后，175,000道尔顿的片段转化为150,000道尔顿区域的片段，该片段与未改变的150,000道尔顿片段一起，其含量似乎与130,000道尔顿的片段相当。这一发现表明，150,000和130,000道尔顿的片段可能起源于不同的亚基链。由于纤溶酶处理过的纤连蛋白经组织蛋白酶B消化后未产生175,000道尔顿的片段，因此得出结论，175,000道尔顿的片段含有连接天然亚基链的链间二硫键。这些结果表明，猪血浆纤连蛋白具有不同的亚基链，这些亚基链由在与肝素的相互作用以及对组织蛋白酶B的敏感性方面存在差异的结构域组成。
