Kirsch T, von der Mark K
Clinical Research Units for Rheumatology, University of Erlangen-Nürnberg, Federal Republic of Germany.
Eur J Biochem. 1991 Mar 28;196(3):575-80. doi: 10.1111/j.1432-1033.1991.tb15852.x.
Type X collagen was extracted with 1 M NaCl and 10 mM dithiothreitol at neutral pH from fetal human growth plate cartilage and purified to homogeneity by gel filtration and anion-exchange chromatography. The purified protein migrates in SDS/polyacrylamide gels with an apparent Mr of 66,000 under reducing conditions, and as a high-Mr oligomer under non-reducing conditions. Purified collagenase digests most of the molecule; pepsin digestion at 4 degrees C decreases the Mr of the monomer to 53,000. A rabbit antiserum was raised against purified human type X collagen; the IgG fraction was specific for this collagen by criteria of ELISA and immunoblotting after absorption with collagen types I, II, VI, IX and XI. Immunohistological studies localized type X collagen exclusively in the zone of hypertrophic and calcifying cartilage.
在中性pH条件下，用1M氯化钠和10mM二硫苏糖醇从人胎儿生长板软骨中提取X型胶原，并通过凝胶过滤和阴离子交换色谱法纯化至同质。纯化后的蛋白质在还原条件下于SDS/聚丙烯酰胺凝胶中迁移，表观分子量为66,000，在非还原条件下则以高分子量寡聚体形式存在。纯化的胶原酶可消化该分子的大部分；4℃下胃蛋白酶消化可使单体的分子量降至53,000。制备了针对纯化的人X型胶原的兔抗血清；通过ELISA和用I、II、VI、IX和XI型胶原吸收后的免疫印迹标准，IgG组分对该胶原具有特异性。免疫组织学研究将X型胶原仅定位在肥大和钙化软骨区域。
