Cole T C, Burkhardt D, Frost L, Ghosh P
Biochim Biophys Acta. 1985 Apr 17;839(2):127-38. doi: 10.1016/0304-4165(85)90029-7.
The high-buoyant-density proteoglycans of the nucleus pulposus and annulus fibrosus of the beagle intervertebral disc have been isolated by CsCl density gradient ultracentrifugation. The sulphated proteoglycans were labelled in vivo with 35SO4, 24 h and 60 days prior to killing. The hydrodynamic size and aggregation of the 24 h, 60 day and resident (from hexuronic acid and hexosamine analysis) proteoglycan subunit populations were determined by Sepharose CL-2B chromatography in the presence or absence of excess hyaluronic acid. The hydrodynamic size of the keratan sulphate-proteoglycan core protein complexes were also determined by Sepharose CL-2B chromatography after chondroitinase ABC digestion of proteoglycans. When initially synthesised (24 h) or after 60 days, the percentage aggregation and hydrodynamic size of the proteoglycans derived from the annulus fibrosus were larger than those present in the nucleus pulposus. Hexosamine, hexuronic and protein determination of the high-buoyant-density fractions showed that the proteoglycans of the nucleus pulposus were richer in chondroitin sulphate than those in the annulus. However there was no difference in Mr of the chondroitin sulphate and keratan sulphate attached to the proteoglycans of the two disc regions, nor were differences detected by HPLC between the proportions of chondroitin 4-sulphate and chondroitin 6-sulphate present in these high-density fractions. In contrast, the low-buoyant-density (1.54 greater than p greater than 1.45) proteoglycan fractions and tissue residues remaining after 4 M GuHCl extraction were found to contain dermatan sulphate, suggesting the presence of a third proteoglycan species possibly associated with the collagen of the fibrocartilagenous matrix.
通过氯化铯密度梯度超速离心法，分离出了比格犬椎间盘髓核和纤维环的高浮力密度蛋白聚糖。在处死前24小时和60天，用35SO4对硫酸化蛋白聚糖进行体内标记。在存在或不存在过量透明质酸的情况下，通过琼脂糖CL-2B色谱法测定了24小时、60天和驻留（根据己糖醛酸和己糖胺分析）蛋白聚糖亚基群体的流体力学大小和聚集情况。在蛋白聚糖经软骨素酶ABC消化后，也通过琼脂糖CL-2B色谱法测定了硫酸角质素-蛋白聚糖核心蛋白复合物的流体力学大小。最初合成时（24小时）或60天后，纤维环来源的蛋白聚糖的聚集百分比和流体力学大小大于髓核中的。高浮力密度级分的己糖胺、己糖醛酸和蛋白质测定表明，髓核的蛋白聚糖比纤维环的富含硫酸软骨素。然而，两个椎间盘区域的蛋白聚糖所连接的硫酸软骨素和硫酸角质素的分子量没有差异，通过高效液相色谱法也未检测到这些高密度级分中硫酸软骨素4-硫酸酯和硫酸软骨素6-硫酸酯比例的差异。相比之下，发现低浮力密度（1.54＞p＞1.45）的蛋白聚糖级分和4M盐酸胍提取后剩余的组织残渣含有硫酸皮肤素，这表明可能存在第三种蛋白聚糖，可能与纤维软骨基质的胶原蛋白有关。
