Purification and N-terminal amino acid sequence of a chondroitin sulphate/dermatan sulphate proteoglycan isolated from intima/media preparations of human aorta.
Purification and N-terminal amino acid sequence of a chondroitin sulphate/dermatan sulphate proteoglycan isolated from intima/media preparations of human aorta.
复制标题
从人主动脉内膜/中膜制备物中分离出的硫酸软骨素/硫酸皮肤素蛋白聚糖的纯化和 N 末端氨基酸序列。
作者：
G. Stöcker;H. Meyer;C. Wagener;H. Greiling
通过Sepharose Q阴离子交换、Sepharose CL-4B大小排除、羟基磷灰石、MonoQ阴离子交换和TSK G 4000 SW大小排除等色谱步骤，从人主动脉内膜/培养基中纯化出一种蛋白多糖（PG）。通过SDS/PAGE用银或二甲基亚甲基蓝直接染色，以及生物素标记样品的Western blots来确定该制剂的纯度。天然PG的电泳迁移率低于20万mr标准蛋白的电泳迁移率。用硫酸软骨素裂解酶ABC处理后，发现了Mr 15000的核心蛋白。与硫酸角蛋白肽相比，糖胺聚糖（GAG）肽的Mr小于24000。GAG链的组成通过硫酸软骨素裂解酶AC/ABC或硫酸软骨素裂解酶AC单独对PG进行不同的酶切，然后对所得双糖进行阴离子交换色谱测定。GAG链由大约三分之一的硫酸皮肤素和三分之二的硫酸软骨素双糖单位组成。20个n端氨基酸的序列与先前报道的从人类发育骨骼中分离的PG I的序列相同[Fisher， Termine & Young (1989) J. Biol]。化学学报，2004,26(4):481 - 476。糖基化位点被分配到位置5和10的丝氨酸残基。结果表明，硫酸软骨素/硫酸皮肤素PG是人主动脉内膜/介质制备中的主要PG，是一种大狼型PG。
A proteoglycan (PG) was purified to homogeneity from intima/media preparations of human aorta specimens by the following chromatographic steps: Sepharose Q anion exchange, Sepharose CL-4B size exclusion, hydroxyapatite, MonoQ anion exchange and TSK G 4000 SW size exclusion. The purity of the preparation was established by SDS/PAGE using direct staining by silver or Dimethylmethylene Blue, as well as by Western blots of biotin-labelled samples. The electrophoretic mobility of the native PG was less than that of a 200,000-Mr standard protein. After treatment with chondroitin sulphate lyase ABC, a core protein of Mr 15,000 was revealed. The Mr of the glycosaminoglycan (GAG) peptides was less than 24,000, by comparison with a keratan sulphate peptide. The composition of the GAG chains was determined by differential digestion of the PG by chondroitin sulphate lyases AC/ABC or chondroitin sulphate lyase AC alone followed by anion-exchange chromatography of the resulting disaccharides. The GAG chains are composed of approximately one-third of dermatan sulphate and two-thirds chondroitin sulphate disaccharide units. The sequence of the 20 N-terminal amino acids is identical with the sequence previously reported for PG I isolated from human developing bone [Fisher, Termine & Young (1989) J. Biol. Chem. 264, 4571-4576]. The assignment of glycosylation sites to the serine residues in positions 5 and 10 was confirmed. The findings indicate that the chondroitin sulphate/dermatan sulphate PG is a major PG in intima/media preparations of human aorta and represents a biglycan-type PG.