Riou J F, Alfandari D, Eppe M, Tacchetti C, Chiquet M, Boucaut J C, Thiery J P, Levi G
Laboratorie de Biologie Expérimentale, CNRS, Université P. et M. Curie, Paris, France.
FEBS Lett. 1991 Feb 25;279(2):346-50. doi: 10.1016/0014-5793(91)80184-5.
We report here the purification of tenascin, an extracellular matrix molecule involved in the control of morphogenesis, from the conditioned medium of the Xenopus XTC cell line. Tenascin was purified by affinity chromatography on a column of the monoclonal antibody mAb TnM1; the molecule eluted from this column has a relative molecular mass of 210 kDa after reduction. Electrophoretic analysis under non-reducing conditions shows that the purified components are oligomeric disulfide-linked complexes which barely enter a 4% polyacrylamide gel. Upon rotary shadowing these molecules appear to possess a central globular domain to which pairs or triplets of arms are attached. Polyclonal antibodies have been raised against purified Xenopus tenascin. They recognise specifically the antigen on Western blots of XTC conditioned medium and adult brain, by immunofluorescence, these antibodies reveal large amounts of tenascin in the secretory vesicles as well as in the extracellular matrix of XTC cells. In the Xenopus tadpole, they stain the developing cartilage, the basal lamina of skin epidermis, myotendinous ligaments and restricted regions of the central nervous system.
我们在此报告从非洲爪蟾XTC细胞系的条件培养基中纯化肌腱蛋白（一种参与形态发生调控的细胞外基质分子）的过程。肌腱蛋白通过在单克隆抗体mAb TnM1柱上进行亲和层析来纯化；从该柱上洗脱下来的分子在还原后相对分子质量为210 kDa。非还原条件下的电泳分析表明，纯化后的组分是寡聚二硫键连接的复合物，几乎不能进入4%的聚丙烯酰胺凝胶。经旋转投影后，这些分子似乎具有一个中央球状结构域，其上连接着成对或三个一组的臂。已制备出针对纯化的非洲爪蟾肌腱蛋白的多克隆抗体。它们在XTC条件培养基和成年脑的蛋白质免疫印迹中能特异性识别抗原，通过免疫荧光法，这些抗体显示分泌小泡以及XTC细胞的细胞外基质中有大量肌腱蛋白。在非洲爪蟾蝌蚪中，它们能对发育中的软骨、皮肤表皮的基膜、肌腱韧带和中枢神经系统的特定区域进行染色。
