Marcum J A, Atha D H, Fritze L M, Nawroth P, Stern D, Rosenberg R D
J Biol Chem. 1986 Jun 5;261(16):7507-17.
Cloned bovine aortic endothelial cells were cultured with [35S]Na2SO4 and proteolyzed extensively with papain. Radiolabeled heparan sulfate was isolated by DEAE-Sephacel chromatography. The mucopolysaccharide was then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate, which bound tightly to the protease inhibitor, represented 0.84% of the mucopolysaccharide mass, accounted for greater than 99% of the initial anticoagulant activity, and exhibited a specific activity of 1.16 USP units/10(6) 35S-cpm. However, the heparan sulfate that interacted minimally with the protease inhibitor constituted greater than 99% of the mucopolysaccharide mass, represented less than 1% of the starting biologic activity, and possessed a specific anticoagulant potency of less than 0.0002 USP unit/10(6) 35S-cpm. An examination of the disaccharide composition of the two populations revealed that the high-affinity heparan sulfate contained a 4-fold or greater amount of GlcA----GlcN-SO3-3-O-SO3 (where GlcA is glucuronic acid), which is a marker for the antithrombin-binding domain of commercial heparin, as compared with the depleted material. Cloned bovine aortic endothelial cells were incubated with [35S]Na2SO4 as well as tritiated amino acids and completely solubilized with 4 M guanidine hydrochloride and detergents. The double-labeled proteoglycans were isolated by DEAE-Sephacel, Sepharose CL-4B, and octyl-Sepharose chromatography. These hydrophobic macromolecules were then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate proteoglycans which bound tightly to the protease inhibitor represented less than 1% of the starting material and exhibited a specific anticoagulant activity as high as 21 USP units/10(6) 35S-cpm, whereas the heparan sulfate proteoglycan that interacted weakly with the protease inhibitor constituted greater than 99% of the starting material and possessed a specific anticoagulant potency as high as 0.02 USP unit/10(6) 35S-cpm. The high-affinity heparan sulfate proteoglycan is responsible for more than 85% of the anticoagulant activity of the cloned bovine aortic endothelial cells. Binding studies conducted with 125I-labeled antithrombin demonstrated that these biologically active proteoglycans are located on the surface of cloned bovine aortic endothelial cells.
将克隆的牛主动脉内皮细胞用[35S]Na2SO4培养，并用木瓜蛋白酶进行广泛的蛋白水解。通过DEAE - Sephacel色谱法分离放射性标记的硫酸乙酰肝素。然后利用固定化抗凝血酶将该粘多糖亲和分离为两个独立的群体。与蛋白酶抑制剂紧密结合的硫酸乙酰肝素占粘多糖质量的0.84%，占初始抗凝血活性的99%以上，比活性为1.16 USP单位/10(6) 35S - 每分钟计数。然而，与蛋白酶抑制剂相互作用最小的硫酸乙酰肝素占粘多糖质量的99%以上，起始生物活性的比例不到1%，且比抗凝血效力小于0.0002 USP单位/10(6) 35S - 每分钟计数。对这两个群体的二糖组成进行检查发现，与耗尽的物质相比，高亲和力硫酸乙酰肝素中GlcA----GlcN - SO3 - 3 - O - SO3（其中GlcA是葡萄糖醛酸）的含量高出4倍或更多，这是商业肝素抗凝血酶结合结构域的一个标志物。将克隆的牛主动脉内皮细胞与[35S]Na2SO4以及氚标记的氨基酸一起孵育，并用4 M盐酸胍和去污剂完全溶解。通过DEAE - Sephacel、琼脂糖CL - 4B和辛基 - 琼脂糖色谱法分离双标记的蛋白聚糖。然后利用固定化抗凝血酶将这些疏水性大分子亲和分离为两个独立的群体。与蛋白酶抑制剂紧密结合的硫酸乙酰肝素蛋白聚糖占起始材料的不到1%，比抗凝血活性高达21 USP单位/10(6) 35S - cpm，而与蛋白酶抑制剂弱相互作用的硫酸乙酰肝素蛋白聚糖占起始材料的99%以上，比抗凝血效力高达0.02 USP单位/10(6) 35S - cpm。高亲和力硫酸乙酰肝素蛋白聚糖占克隆的牛主动脉内皮细胞抗凝血活性的85%以上。用125I标记的抗凝血酶进行的结合研究表明，这些具有生物活性的蛋白聚糖位于克隆的牛主动脉内皮细胞表面。
