Björnsson S, Heinegård D
Biochem J. 1981 Oct 1;199(1):17-29. doi: 10.1042/bj1990017.
The assembly of proteoglycan aggregates in chondrocyte cell cultures was examined in pulse-chase experiments with the use of [35S]sulphate for labelling. Rate-zonal centrifugation in linear sucrose density gradients (10-50%, w/v) was used to separate the aggregated proteoglycans from monomers and to assess the size of the newly formed aggregates. The proportion of aggregates stabilized by link protein was assessed by competition with added exogenous aggregate components. The capacity of the proteoglycans synthesized in culture to compete with exogenous nasal-cartilage proteoglycans for binding was studied in dissociation-reassociation experiments. The results were as follows. (a) The proteoglycan monomers and the hyaluronic acid are exported separately and combined extracellularly. (b) The size of the aggregates increases gradually with time as the proportion of monomers bound to hyaluronic acid increases. (c) All of the aggregates present at a particular time appear to be link-stabilized and therefore not dissociated by added excess of nasal-cartilage proteoglycan monomer or hyaluronic acid oligomers. (d) The free monomer is apparently present as a complex with link protein. The monomer-link complexes are then aggregated to the hyaluronic acid. (e) The aggregates synthesized in vitro and the nasal-cartilage aggregates differ when tested for link-stabilization by incubation at low pH. The aggregates synthesized in vitro were completely dissociated whereas the cartilage proteoglycans remained aggregated. The results obtained from dissociation-reassociation experiments performed at low pH indicate that the proteoglycan monomer synthesized in vitro does not bind the hyaluronic acid or the link protein as strongly as does the nasal-cartilage monomer.
利用[35S]硫酸盐进行标记，通过脉冲追踪实验研究软骨细胞培养物中蛋白聚糖聚集体的组装。采用线性蔗糖密度梯度（10 - 50%，w/v）进行速率区带离心，以将聚集的蛋白聚糖与单体分离，并评估新形成聚集体的大小。通过与添加的外源聚集体成分竞争，评估由连接蛋白稳定的聚集体比例。在解离 - 重聚实验中，研究培养物中合成的蛋白聚糖与外源鼻软骨蛋白聚糖竞争结合的能力。结果如下：（a）蛋白聚糖单体和透明质酸分别输出并在细胞外结合。（b）随着与透明质酸结合的单体比例增加，聚集体的大小随时间逐渐增加。（c）在特定时间存在的所有聚集体似乎都由连接蛋白稳定，因此不会因添加过量的鼻软骨蛋白聚糖单体或透明质酸寡聚物而解离。（d）游离单体显然以与连接蛋白的复合物形式存在。单体 - 连接蛋白复合物随后聚集到透明质酸上。（e）当在低pH下孵育测试连接蛋白稳定性时，体外合成的聚集体和鼻软骨聚集体有所不同。体外合成的聚集体完全解离，而软骨蛋白聚糖仍保持聚集状态。在低pH下进行的解离 - 重聚实验结果表明，体外合成的蛋白聚糖单体与透明质酸或连接蛋白的结合不如鼻软骨单体紧密。
