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

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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.