Further characterization of the binding of fibronectin to gelatin reveals the presence of different binding interactions.
The specific interaction between fibronectin and collagen has permitted the isolation of fibronectin from plasma using gelatin-Sepharose affinity matrices. In this study we obtained evidence suggesting the presence of an additional high affinity binding interaction between fibronectin and collagen. The following information supports this idea: (i) Successive isolations of fibronectin using the same regenerated gelatin matrix resulted in progressively increased yields of fibronectin, thus indicating the progressive occupation of high affinity fibronectin-binding sites within collagen. (ii) This tightly bound fibronectin could not be eluted from gelatin-Sepharose matrices with strong denaturing agents, such as 8.0 M urea or 6.0 M guanidium chloride. (iii) The fibronectin-occupied matrices supported fibroblast adhesion and spreading, indicating that the amount of tightly bound fibronectin was quantitatively sufficient for this function, and that the fibronectin that bound with high affinity retained its biologically active conformation. (iv) Two fibronectin-derived fragments, CB52kDa and T55 kDa, selectively bound to fresh gelatin-Sepharose but not to gelatin-Sepharose previously employed to purify fibronectin, suggesting that these fragments recognize only the high affinity binding sites in gelatin. N-terminal amino acid sequence analysis of T55 kDa showed that it contained the previously described collagen-binding domain and included the repeat III-1 of fibronectin. These results support the existence of multiple fibronectin-binding sites in collagen and implicate a high affinity site(s) with a propensity for tenacious binding.
Garcia-Pardo
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A
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
Gold
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D001665:Binding Sites; D002448:Cell Adhesion; D003094:Collagen / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D011485:Protein Binding
D005353:Fibronectins; D005780:Gelatin; D003094:Collagen