Flannery C R, Urbanek P J, Sandy J D
Department of Orthopaedics, Brown University, Rhode Island Hospital, Providence.
J Orthop Res. 1990 Jan;8(1):78-85. doi: 10.1002/jor.1100080110.
We have examined extracts of articular cartilage from rabbits aged 3-100 weeks for evidence of age-related changes in the structure and content of link protein (LP) in this tissue, with the following findings: (a) Two major molecular weight forms of LP were seen on SDS-PAGE (41 and 48 kDa) and the proportion of these changed markedly with age. The 48 kDa species was predominant in young animals (representing about 78% of the total LP at 5 weeks) whereas the 41 kDa species increased in amount with age (representing 35% of the total LP at 100 weeks). A minor form of about 43 kDa, representing less than 20% of the total, was present only during the growth phase. A small amount of fragmented link protein (less than 5% of the total) of about 25-30 kDa was present in samples from mature and aged rabbits only. (b) The quantitation of LP in guanidinium: HCl extracts of cartilage, by radioimmunoassay with monoclonal antibody 8-A-4, was markedly influenced by the conditions of preparation and pretreatment of samples. Assays of dialyzed guanidine extracts following treatment at 80 degrees C for 15 min in 0.025% (w/v) SDS indicated that immature and mature cartilage contains about 50 and 180 micrograms of LP/g of tissue, respectively. On the other hand, assays following treatment at 100 degrees C for 20 min in 0.1% (w/v) SDS suggested that rabbit cartilage contains about 300 micrograms of LP/g of tissue at all ages; finally, assay of CsCl purified proteoglycan samples under these conditions indicated a content of about 500 micrograms of LP/g at all ages. (c) Calculations based on the analysis of proteoglycan preparations for aggregating monomer and link protein suggest that a LP:aggregating monomer molar ratio of about 0.9 is maintained in the articular cartilage throughout maturation and aging in the rabbit.
我们检测了3至100周龄兔子的关节软骨提取物，以寻找该组织中连接蛋白（LP）结构和含量随年龄变化的证据，结果如下：（a）在SDS-PAGE上可见LP的两种主要分子量形式（41和48 kDa），且这些形式的比例随年龄显著变化。48 kDa的种类在幼龄动物中占主导（5周龄时占总LP的约78%），而41 kDa的种类数量随年龄增加（100周龄时占总LP的35%）。一种约43 kDa的次要形式，占总量不到20%，仅在生长阶段存在。仅在成熟和老龄兔子的样本中存在少量约25 - 30 kDa的片段化连接蛋白（占总量不到5%）。（b）用单克隆抗体8-A-4通过放射免疫测定法对软骨的盐酸胍提取物中的LP进行定量时，样本的制备和预处理条件对其有显著影响。在0.025%（w/v）SDS中于80℃处理15分钟后对透析后的胍提取物进行测定表明，未成熟和成熟软骨分别含有约50和180微克LP/克组织。另一方面，在0.1%（w/v）SDS中于100℃处理20分钟后进行测定表明，所有年龄段的兔软骨含有约300微克LP/克组织；最后，在这些条件下对CsCl纯化的蛋白聚糖样本进行测定表明，所有年龄段的LP含量约为500微克/克。（c）基于对用于聚集单体和连接蛋白的蛋白聚糖制剂的分析计算表明，在兔子整个成熟和衰老过程中，关节软骨中LP与聚集单体的摩尔比约为0.9得以维持。
