Isolation and characterization of a link protein from bovine aorta proteoglycan aggregate.
Isolation and characterization of a link protein from bovine aorta proteoglycan aggregate.
复制标题
从牛主动脉蛋白聚糖聚集体中分离和表征连接蛋白。
DOI：
10.1016/0304-4165(85)90188-6
复制
发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Berenson,GS
中科院分区：
文献类型：
--
作者：
Vijayagopal,P;Radhakrishnamurthy,B;Srinivasan,SR;Berenson,GS
以0.5M盐酸胍为萃取剂，在有酶抑制剂存在下，从牛主动脉中提取蛋白多糖聚集体，并用等密度氯化铯离心法进行纯化。梯度的底部五分之二(A1)含有30%的聚集形式的蛋白多糖。其中蛋白质14.8%，己糖酸20.4%，透明质酸、硫酸皮肤素、硫酸软骨素按18：18：69比例混合而成。通过离解CsC l等密度离心法，从最低的五分之二中分离出一个含有连接蛋白的组分。用Sephadex G-200在4M盐酸胍存在下，通过Sephadex G-200层析纯化了漂浮到梯度顶端五分之一的连接蛋白。在SDS-聚丙烯酰胺凝胶电泳法中呈单一条带移动，相对分子质量为49000。LINK蛋白的氨基酸组成与软骨LINK蛋白相似，但与蛋白多糖单体的蛋白核心的氨基酸组成有显著差异。连接蛋白的中性糖含量为干重的3.5%。半乳糖、甘露糖和岩藻糖分别占总中性糖的21%、62%和16%。在聚集研究中，发现该连接蛋白与蛋白多糖单体和透明质酸相互作用。由透明质酸衍生的低聚糖降低了蛋白多糖和透明质酸的无连接蛋白聚集体的粘度，但不降低连接稳定的聚集体的粘度，表明连接蛋白增加了蛋白多糖聚集体的稳定性。
Proteoglycan aggregates were isolated from bovine aorta by extraction with 0.5 M guanidine hydrochloride in the presence of proteinase inhibitors and purified by isopycnic CsCl centrifugation. The bottom two-fifths (A1) of the gradient contained 30% of proteoglycans in the aggregated form. The aggregate had 14.8% protein and 20.4% hexuronic acid with hyaluronic acid, dermatan sulfate and chondroitin sulfates in a proportion of 18:18:69. A link protein-containing fraction was isolated from the bottom two-fifths by dissociative CsCl isopycnic centrifugation. The link protein that floated to the top one-fifth of the gradient was purified by chromatography on Sephadex G-200 in the presence of 4 M guanidine hydrochloride. It moved as a single band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 49 000. The amino acid composition of link protein resembled that of link protein from cartilage, but was strikingly different from that of the protein core of the proteoglycan monomer. The neutral sugar content of link protein was 3.5% of dry weight. Galactose, mannose and fucose constituted 21, 62 and 16%, respectively of the total neutral sugars. In aggregation studies the link protein was found to interact with both proteoglycan monomer and hyaluronic acid. Oligosaccharides derived from hyaluronic acid decreased the viscosity of link protein-free aggregates of proteoglycan and hyaluronic acid but not of link-stabilized aggregates, demonstrating that the link protein increases the stability of proteoglycan aggregates.