Molecular chractrerization of cell aggregation factor(s) in celluar fibronectin prepatration - A possibility of a determinant chondrogene

从鸡胚胎成纤维细胞制备的细胞纤维连接蛋白具有促进鸡胚胎肢芽间充质细胞体外聚集和随后聚集的细胞的软骨形成的活性物质(S)。经SepharoseCL-2b柱层析分离，发现CCFN的低分子量组分有活性，在SDS-聚丙烯酰胺凝胶电泳法上，在Mr=15,000附近有少量糖蛋白条带。Kasai等人(Teikyo University)证明了在对照胚胎中存在两种β-半乳糖苷结合凝集素，其MR分别为16,000和14,000。这些凝集素及其特异性抗体用于观察CCFN中的活性物质(S)是否与这些凝集素相同。SDS-PAGE和随后使用这些抗体的免疫印迹表明，CCFN中不仅存在MR=16,000和MR=14,000的凝集素，而且在22-23期的对照胚胎肢芽的洗涤剂提取物中也存在MR=16,000的LC。在肢芽间充质细胞培养液中添加适量腐烂的凝集素，可促进细胞聚集。两种抗体中的任何一种同时加入CCFN均可中和诱导的细胞聚集。此外，在不含CCFN的培养液中加入抗体后，慢速细胞聚集完全消失，这可能是由于选择的内源性合成所致。因此，CCFN中的活性底物可能是由污染物引起的，MR=16,000和14,000β-半乳糖苷结合凝集素，前者存在于肢芽中，在肢芽间充质凝聚过程中对细胞聚集起重要作用。

Cellular fibronection prepared from chick embryonic fibroblast cells (CCFN) has been shown to have the active suvstance(s) to promote in vitro aggregation of chick embryonic limb bud mesencymal cells and subsequent chondrogrmesis of the aggregated cells. When fractionated by Sepharose CL 2b column chromatography, we found the activity in the lower molecular weight fraction of CCFN which gave a few glycoprotein bands around Mr=15,000 on SDS-polyacrylamide gel electrohoresis. Kasai et al(Teikyo University) have demonstrated the existence of tw kinds of beta-galactoside-binding lectins having Mr=16,000 and Mr=14,000, respectively, in check embryo. These lectins and their specific antibodies were served to see whether the active substance(s)in CCFN is identical with these lectine ot not. SDS-polyacrylamide gel electrophoresis and subsequent immunoblotting using these antibodies suggested the presence of not only both the Mr=16,000 and Mr=14,000 lectins in CCFN but also the Mr=16,000 lCtin in the detergent extract of check embryo limb buds at stages 22-23. The difect addition of the putified lectine to the culture medium of the limb bud mesenchymal cells prometed the cell aggregation. The simultaneous addition of either the antibodies with CCFN neutralized the induced cell aggregation. Furthermore, the addition of the antibodies to the culture without CCFN brought about the complete disappearance of the slowly going cell aggregation which was probably due to the endogenous synthesis of the lection. Therefore, it is likely that the active substrances in CCFN is caused by the contaminants, Mr=16,000 and 14,000 beta-galactoside-binding lectins and that the former lectin exists in the limb buds and plays an importanr role in the cell aggregation during limb bud mesenchymal condensation.