Choi H U, Tang L H, Johnson T L, Rosenberg L
J Biol Chem. 1985 Oct 25;260(24):13370-6.
Two forms of link protein, 46 and 51 kDa, are present in proteoglycan aggregates from both bovine nasal and bovine articular cartilages. Studies reported here show that the link proteins bind to concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin, soybean agglutinin, and wheat germ agglutinin lectins. When the link proteins are eluted from these lectins with appropriate competing sugars, the 46- and the 51-kDa link proteins elute together and no separation is achieved. However, when the link proteins bound to wheat germ agglutinin are eluted with a 0 to 4 M guanidine hydrochloride linear gradient, a good separation of the 46- and 51-kDa link proteins is achieved. Wheat germ agglutinin affinity chromatography has been used on a preparative scale to isolate the 51-kDa link protein from mature bovine articular cartilage to homogeneity, in amounts sufficient to examine its effect on proteoglycan aggregate size and stability in sedimentation velocity studies. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronate in the absence of link protein, in the presence of both 46- and 51-kDa link proteins, and in the presence of the individual 51-kDa link protein. The sizes of the aggregates were compared in terms of sedimentation coefficients (s(0)20). The stability of the aggregates was compared in terms of the per cent aggregate present at pH 7 and 5. At pH 7, the sedimentation coefficients (s(0)20) of link-free aggregates, aggregates formed with both link proteins, and aggregates formed with 51-kDa link protein were 72, 93, and 112 S, respectively. Thus, the 51-kDa link protein has a pronounced effect on aggregate size. The link-free aggregate was grossly unstable, and only 36% aggregate was present at pH 5. The aggregate formed with both link proteins was effectively stabilized against dissociation and 79% aggregate was present at pH 5. The aggregate formed with 51-kDa link protein was not effectively stabilized against dissociation, and only 60% aggregate was present at pH 5. Thus, despite its pronounced effect on aggregate size, the 51-kDa link protein does not effectively stabilize the proteoglycan aggregate against dissociation. These results suggest that the 51-kDa link protein may selectively increase aggregate size, while the 46-kDa link protein may be required to effectively stabilize the proteoglycan aggregate against dissociation.
牛鼻软骨和牛关节软骨的蛋白聚糖聚集体中存在两种形式的连接蛋白，分子量分别为46 kDa和51 kDa。本文报道的研究表明，连接蛋白能与伴刀豆球蛋白A、小扁豆凝集素、蓖麻凝集素、大豆凝集素和麦胚凝集素结合。当用适当的竞争性糖类从这些凝集素上洗脱连接蛋白时，46 kDa和51 kDa的连接蛋白一起洗脱，无法分离。然而，当用0至4 M盐酸胍线性梯度洗脱与麦胚凝集素结合的连接蛋白时，46 kDa和51 kDa的连接蛋白能得到很好的分离。麦胚凝集素亲和层析已被用于从成熟牛关节软骨中大规模分离51 kDa的连接蛋白，得到的量足以在沉降速度研究中检测其对蛋白聚糖聚集体大小和稳定性的影响。在无连接蛋白、同时存在46 kDa和51 kDa连接蛋白以及单独存在51 kDa连接蛋白的情况下，从蛋白聚糖单体和透明质酸重新组装蛋白聚糖聚集体。根据沉降系数（s(0)20）比较聚集体的大小。根据在pH 7和pH 5时聚集体所占的百分比比较聚集体的稳定性。在pH 7时，无连接蛋白的聚集体、由两种连接蛋白形成的聚集体以及由51 kDa连接蛋白形成的聚集体的沉降系数（s(0)20）分别为72 S、93 S和112 S。因此，51 kDa的连接蛋白对聚集体大小有显著影响。无连接蛋白的聚集体非常不稳定，在pH 5时只有36%的聚集体存在。由两种连接蛋白形成的聚集体能有效稳定，防止解离，在pH 5时有79%的聚集体存在。由51 kDa连接蛋白形成的聚集体不能有效稳定，防止解离，在pH 5时只有60%的聚集体存在。因此，尽管51 kDa的连接蛋白对聚集体大小有显著影响，但它不能有效稳定蛋白聚糖聚集体防止解离。这些结果表明，51 kDa的连接蛋白可能选择性地增加聚集体大小，而46 kDa的连接蛋白可能是有效稳定蛋白聚糖聚集体防止解离所必需的。
