Characterization of proteoglycans isolated from associative extracts of human articular cartilage.
Characterization of proteoglycans isolated from associative extracts of human articular cartilage.
复制标题
从人关节软骨联合提取物中分离出的蛋白多糖的表征。
作者：
V. Vilím;A. Fosang
在Dulbecco's PBS的联合条件下，提取正常年轻人关节软骨总蛋白多糖含量的约10%。用Q-Sepharose层析分离得到蛋白多糖，用凝胶层析分离，用梯度凝胶SDS/PAGE和免疫印迹对其进行表征。鉴定出3种小蛋白多糖、2个聚集蛋白主要居群及其较小片段居群。聚集蛋白的主要群体包含硫酸软骨素链，全部或部分的n端G1和G2结构域，因此，完整的硫酸角蛋白结构域。通过梯度SDS/PAGE估计较大的群体的分子质量约为。600kda或更大。第二居群的表观分子质量约为。300 - 600 kDa。从这些蛋白聚糖群体中提取的核心蛋白在SDS/PAGE上被分离成几个在120到大约范围内的带簇。360 kDa。该提取物还含有更小的片段，这些片段缺乏硫酸软骨素，但与针对硫酸角蛋白的抗体和存在于聚集蛋白G2结构域的表位反应。G2结构域的存在表明聚集蛋白核心蛋白在其硫酸软骨素结构域内发生了广泛的裂解。这些发现表明，聚集蛋白的断裂可能发生在年轻个体正常关节软骨的体内。联合提取物还含有decorin、biglycan和纤维调节素。用凝胶层析法从聚集蛋白中分离出来，用免疫检测法鉴定。
Approx. 10% of the total proteoglycan content of normal young human articular cartilage was extracted under associative conditions with Dulbecco's PBS. Proteoglycans isolated from the extract by Q-Sepharose chromatography were separated by gel chromatography and characterized by gradient gel SDS/PAGE and immunoblotting. Three species of small proteoglycans, two main populations of aggrecan and a population of its smaller fragments were identified. The major populations of aggrecan contained chondroitin sulphate chains, all or part of the N-terminal G1 and G2 domains and, therefore, intact keratan sulphate domains. The larger population was estimated by gradient SDS/PAGE to have a molecular mass of approx. 600 kDa or greater. The second population had an apparent molecular mass of approx. 300-600 kDa. Core proteins derived from these populations of proteoglycans separated on SDS/PAGE into several clusters of bands in the range from 120 to approx. 360 kDa. The extract further contained smaller fragments which lacked chondroitin sulphate but reacted with antibodies against keratan sulphate, and against epitopes present in the G2 domain of aggrecan. The presence of the G2 domain in a broad range of populations of decreasing size indicated extensive cleavage of the aggrecan core protein within its chondroitin sulphate domain. These findings suggest that fragmentation of aggrecan probably occurs in vivo in normal articular cartilage of young individuals. Associative extracts also contained decorin, biglycan and fibromodulin. These were resolved from aggrecan by gel chromatography and identified by immunodetection.