Isolation of Endothelial Cell Proteoheparan Sulfate and Preparationof Its Antibody

在分离内皮细胞硫酸乙酰肝素的过程中，发现不同的试剂(&lt；β&gt；-D-木糖苷、正丁酸酯)能改变细胞相关蛋白乙酰肝素的性质。内皮细胞培养与-lt；β-D-木糖苷的孵育导致抗凝血酶III结合和细胞表面硫酸乙酰肝素的生物合成平行减少。正丁酸引起结合量增加，但具有细胞毒性。&lt；β&D-木糖苷不影响细胞生长。而细胞表面硫酸乙酰肝素的大小不受木糖苷处理的影响，在木糖苷存在的情况下，它的净负电荷略少，与抗凝血酶III高亲和力的分子比例显著减少。另一方面，在木糖的存在下，培养上清液中硫酸软骨素的分泌显著增加，而游离乙酰肝素硫酸盐链的分泌增加较少。这些结果表明，-lt；β-D-木糖苷导致细胞表面相关的硫酸乙酰肝素的产量下降和一些细微的结构变化，这可能是抗凝血酶III的结合部位。此外，更重要的是，木苷处理细胞的培养液中硫酸肝素含量的增加表明，该系统可能为该化合物的分离提供一个潜在的有用来源。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.