title: Type I collagen contains at least 14 cryptic fibronectin binding sites of similar affinity.
abstract: There is uncertainty in the literature regarding the number and location of fibronectin binding sites on denatured collagen. Although most attention has focused on a single site near the collagenase-sensitive region of each alpha chain, there is evidence for additional sites in other regions. We treated bovine type I collagen with cyanogen bromide, labeled the resulting mixture with fluorescein, and separated the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent bands were excised from the gel and dialyzed exhaustively to remove detergent. Titration of eight distinct fluorescent-labeled fragments with the 42-kDa gelatin-binding fragment of fibronectin caused increases in anisotropy that were fully reversible with unlabeled gelatin. By fitting the dose responses it was possible to calculate apparent K(d)'s whose values ranged between 1 and 4 microM. The largest fragment, alpha(2)-CB3,5, composing about 2/3 of the alpha(2) chain, when further digested with endoproteinase Lys-C, yielded at least three additional subfragments that also bound with similar affinities. Thus, there appear to be at least 14 distinct fibronectin binding sites of similar affinity in bovine type I collagen, five on each of the alpha(1) chains and four on the alpha(2) chain. Experiments with several synthetic peptides failed to reveal the exact nature of the binding site.
authors.affiliation: Department of Biochemistry, American Red Cross Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855, USA. ingham@usa.redcross.org
authors.forename: K C
authors.initials: KC
authors.lastname: Ingham
authors.forename: S A
authors.initials: SA
authors.lastname: Brew
authors.forename: M
authors.initials: M
authors.lastname: Migliorini
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D024042:Collagen Type I / Q000737:chemistry* / Q000378:metabolism*; D003488:Cyanogen Bromide; D005353:Fibronectins / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
chemical_list: D024042:Collagen Type I; D005353:Fibronectins; D010446:Peptide Fragments; D012967:Sodium Dodecyl Sulfate; D005780:Gelatin; D003488:Cyanogen Bromide