Lark M W, Culp L A
J Biol Chem. 1984 Jun 10;259(11):6773-82.
Both newly formed and long-term culture-generated substratum adhesion sites, generated by EGTA-mediated detachment of Balb/c SVT2 cells, were extracted with an eta-octyl-beta-D-glucopyranoside buffer containing salt and several protease inhibitors under conditions which result in maximal solubilization of the sulfate-radiolabeled proteoglycans. Because of the functional importance of heparan sulfate proteoglycans in the fibronectin-dependent cell-substratum adhesion processes of these cells, these proteoglycans were fractionated on affinity columns of octyl-Sepharose or of the heparan sulfate-binding proteins platelet factor 4 or plasma fibronectin. These affinity matrices resolved a number of both binding and nonbinding classes of heparan sulfate proteoglycan from both types of adhesion sites. In particular, the platelet factor 4 column could resolve several proteoglycans with differing binding affinities. Approximately twice as much heparan sulfate proteoglycan from newly formed sites bound to all three matrices as proteoglycan from longterm sites. The proteoglycan which bound to one matrix was then tested for binding to a second matrix; this approach resolved a number of biochemically distinct species. For example, one-half of the fibronectin-Sepharose-binding fraction from the long-term sites could also bind to platelet factor 4-Sepharose; however, over 90% of the fibronectin-binding fraction from newly formed sites could bind to platelet factor 4. A major portion of the octyl-Sepharose-binding fractions of the original extracts could bind to fibronectin-Sepharose. These studies indicate that some of these proteoglycans have overlapping affinities for fibronectin, platelet factor 4, and octyl-Sepharose and that a portion of the heparan sulfate proteoglycan from these adhesion sites cannot bind to any of these affinity matrices. These results are discussed with regard to the functional significance of these various heparan sulfate proteoglycans in mediating adhesion to extracellular matrices containing fibronectin or platelet factor 4.
通过EGTA介导Balb/c SVT2细胞脱离而产生的新形成的和长期培养产生的基质粘附位点，在能使硫酸根放射性标记的蛋白聚糖最大程度溶解的条件下，用含有盐和几种蛋白酶抑制剂的η-辛基-β-D-吡喃葡萄糖苷缓冲液进行提取。由于硫酸乙酰肝素蛋白聚糖在这些细胞的纤连蛋白依赖性细胞-基质粘附过程中具有功能重要性，这些蛋白聚糖在辛基琼脂糖或硫酸乙酰肝素结合蛋白血小板因子4或血浆纤连蛋白的亲和柱上进行分级分离。这些亲和基质从两种类型的粘附位点分离出了许多结合和非结合类别的硫酸乙酰肝素蛋白聚糖。特别是，血小板因子4柱可以分离出几种具有不同结合亲和力的蛋白聚糖。来自新形成位点的硫酸乙酰肝素蛋白聚糖与所有三种基质结合的量大约是来自长期位点的蛋白聚糖的两倍。然后测试与一种基质结合的蛋白聚糖与第二种基质的结合情况；这种方法分离出了许多生物化学上不同的种类。例如，来自长期位点的纤连蛋白-琼脂糖结合部分的一半也可以与血小板因子4-琼脂糖结合；然而，来自新形成位点的纤连蛋白结合部分的90%以上可以与血小板因子4结合。原始提取物中辛基琼脂糖结合部分的一大部分可以与纤连蛋白-琼脂糖结合。这些研究表明，其中一些蛋白聚糖对纤连蛋白、血小板因子4和辛基琼脂糖具有重叠的亲和力，并且来自这些粘附位点的一部分硫酸乙酰肝素蛋白聚糖不能与任何这些亲和基质结合。讨论了这些不同的硫酸乙酰肝素蛋白聚糖在介导与含有纤连蛋白或血小板因子4的细胞外基质粘附方面的功能意义。
