Isolation of Endothelial Cell Proteoheparan Sulfate and Preparationof Its Antibody

在内皮细胞硫酸肝素的分离过程中，发现多种药物（< β - >- d -木糖苷，正丁酸）可以改变细胞相关的硫酸肝素蛋白的性质。内皮细胞与< β - >- d -木糖苷孵育导致抗凝血酶III结合的平行减少和细胞表面硫酸肝素的生物合成。正丁酸盐引起结合增加，但具有细胞毒性。< β >- d -木糖甙对细胞生长无影响。虽然木糖甙处理没有改变细胞表面硫酸肝素的大小，但在木糖甙的存在下，它的净负电荷略少，对抗凝血酶III具有高亲和力的分子比例显着降低。另一方面，木糖苷存在时，培养基中硫酸软骨素链的分泌量明显增加，同时游离硫酸肝素链的分泌量也有较小的增加。这些结果表明，< β - >- d -木糖苷导致细胞表面相关硫酸肝素的产生减少以及一些微妙的结构改变，硫酸肝素可能作为抗凝血酶III的结合位点。此外，更重要的是，从糖苷处理的细胞培养基中增加硫酸肝素的量表明，该系统可能为分离该化合物提供潜在的有用来源。我们正朝着这个方向继续这个项目。

In the course of isolation of endothelial cell heparan sulfate, various agents (<beta>-D-xyloside, n-butyrate) were found to alter the property of cell-associated proteoheparan sulfate. Incubation of endothelial cell cultures with <beta>-D-xyloside resulted in a parallel reduction of antithrombin III binding and biosynthesis of cell surface heparan sulfate. N-butyrate caused the increase in the binding, but was cytotoxic. <beta>-D-xyloside did not affect the cellular growth. Whereas the size of cell surface heparan sulfate was not altered by xyloside treatment, it apperaed to have slightly less net negative charge and a significantly reduced proportion of the molecule with high affinity for antithrombin III in the presence of xyloside. On the other hand, secretion of chondroitin sulfate chains into the medium was markedly increased in the presence of xyloside, accompanied by a smaller increase in secretion of free heparan sulfate chains. These results suggest that <beta>-D-xyloside caused a decrease in production as well as some subtle structural alterations of cell-surface-associated heparan sulfate, which could serve as binding sites for antithrombin III. Furthermore, more importantly increased amounts of heparan sulfate in the medium from xyoside-treated cells suggest that this system may provide a potentially useful source for the isolation of this compound. We are continuing this project in this direction.