Abstract
The interactions of plasma fibronectin with alpha chains or cyanogen bromide fragments of collagen types I and II have been studied using a variety of techniques. Affinity chromatography of cyanogen bromide-cleaved type II collagen on immobilized fibronectin revealed the binding of cyanogen bromide fragment CB12 in addition to the previously characterized CB10. Using fluorescence polarization, we analyzed the interaction between the collagen peptides and fluorescein isothiocyanate-labeled 42-kDa gelatin-binding fragment of fibronectin in solution. Dissociation constants for the binding of CB10 and CB12 to the fibronectin fragment were calculated as 0.38 and 0.94 microM, respectively, indicating a lower affinity for the uncharacterized site. However, as with CB10, CB12 was able to compete effectively with the intact alpha chain for bindinng to fibronectin. Additionally, both CB10 and CB12 absorbed to tissue culture surfaces were each able to support fibronectin-dependent cell adhesion. Finally, the regions of alpha 2(I) homologous to CB12 and CB10 were found to be active in fibronectin binding, demonstrating the presence of two fibronectin-binding regions in this collagen chain.
Highlights
Fibronectin as the primary treatment served as the positive control and allowed for the attachment of approximately 80
In this study we have demonstrated the presence of a second fibronectin-binding site in al(B) located in peptide CB12 originating near the NH*-terminal region of the chain
Homologous domain in a2(1) had fibronectin-binding activity, whereas a second fibronectin-binding peptide was not detected among the al(I) cleavage products in accordance with previous reports (Kleinman et al, 1976)
Summary
01 chains and cyanogen bromide fragments of type II collagen were isolated from bovine nasal senta as described nreviouslv (Butler et al, 1976). The peptides geierated were fractibnated b; gel filtration on a Superose licolumn (Pharmacia LKB Biotechnology) eluted with 2 M guanidine, 0.05 M. Individual fragments were isolated by reversed phase high-pressure liquid chromatography (HPLC)’ on a-Vydac Cl, PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; DME, Dulbecco’s modified Eagle’s medium; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; SDS-. The identities of the purified fragments were confirmed by amino acid analysis and NHs-terminal sequencing. These were performed by the protein analysis core facilities of the Arteriosclerosis
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