Fellini S A, Kimura J H, Hascall V C
J Biol Chem. 1981 Aug 10;256(15):7883-9.
The population of proteoglycan monomers in aggregates was purified from chondrocyte cultures after labeling with either [3H]serine and [35S]methionine or [3H]serine and [35S]sulfate. Digestion of the monomers labeled with [3H]serine and [35S]methionine with trypsin indicated that serine was enriched (approximately 80%) in the chondroitin sulfate attachment region of the core protein, while methionine was enriched (approximately 60%) in the hyaluronic acid-binding region. Sepharose CL-2B chromatography and velocity gradients were used to isolate monomer subfractions which differed in molecular size. The 3H/35S ratios for the subfractions from monomers labeled with [3H]serine and [35S]methionine were nearly constant, indicating that the core protein lengths were constant regardless of the size of the monomer. Conversely, the 3H/35S ratios for subfractions of monomers labeled with [3H]serine and [35S]sulfate increased significantly with decreasing sizes. Chondroitin sulfate chains in subfractions were released by alkaline borohydride treatment. The mean molecular weights of the chondroitin sulfate chains decreased from approximately 18,500 in the largest subclass of monomers to approximately 12,500 in the smallest. [3H]Serine-labeled monomer subfractions were digested with papain to determine the proportion of serine residues substituted with chondroitin sulfate. The distribution of label was constant for greater than 90% of the monomers from the largest to the smallest. The results indicate that, for at least 90% of the newly synthesized monomers that are able to aggregate, variation in chondroitin sulfate chain size is the only contributing factor to polydispersity in their molecular size.
在用[3H]丝氨酸和[35S]甲硫氨酸或[3H]丝氨酸和[35S]硫酸盐标记后，从软骨细胞培养物中纯化聚集物中的蛋白聚糖单体。用胰蛋白酶消化用[3H]丝氨酸和[35S]甲硫氨酸标记的单体表明，丝氨酸在核心蛋白的硫酸软骨素附着区域富集（约80%），而甲硫氨酸在透明质酸结合区域富集（约60%）。使用琼脂糖CL-2B色谱法和速度梯度来分离分子大小不同的单体亚组分。用[3H]丝氨酸和[35S]甲硫氨酸标记的单体的亚组分的3H/35S比值几乎恒定，表明无论单体大小如何，核心蛋白长度是恒定的。相反，用[3H]丝氨酸和[35S]硫酸盐标记的单体亚组分的3H/35S比值随着大小的减小而显著增加。通过碱性硼氢化钠处理释放亚组分中的硫酸软骨素链。硫酸软骨素链的平均分子量从最大单体亚类中的约18,500降至最小单体亚类中的约12,500。用木瓜蛋白酶消化[3H]丝氨酸标记的单体亚组分，以确定被硫酸软骨素取代的丝氨酸残基的比例。对于从最大到最小的超过90%的单体，标记的分布是恒定的。结果表明，对于至少90%能够聚集的新合成单体，硫酸软骨素链大小的变化是其分子大小多分散性的唯一影响因素。
