Further localization of the gelatin-binding determinants within fibronectin. Active fragments devoid of type II homologous repeat modules.
Digestion of a 42-kDa gelatin-binding fragment (GBF) of fibronectin with pepsin followed by affinity chromatography on gelatin-Sepharose produces three fractions, a drop-through non-binding fraction, a retarded fraction that is dominated by a 13-kDa fragment whose NH2 terminus is identical to that of 42-kDa GBF, and a binding fraction that contains a homogeneous fragment of apparent mass 21 kDa with an NH2 terminus corresponding to Arg484. This 21-kDa GBF binds repeatedly to gelatin-Sepharose, eluting near 2.6 M in a urea gradient. It also binds in the fluid phase to a fluorescent-labeled collagen peptide with Kd = 10 microM and inhibits the binding of 42-kDa GBF to the same peptide with KI = 7.3 microM. Thus, major gelatin-binding determinants of fibronectin are located within a 21-kDa region that contains two type I homologous "finger" modules and is devoid of the type II "kringle-like" modules that were previously thought to be essential for this activity.
Ingham
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KC
Biochemistry Laboratory, American Red Cross Biomedical Research & Development, Rockville, Maryland 20855.
Brew
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SA
Migliorini
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MM
D000595:Amino Acid Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D003094:Collagen / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005780:Gelatin / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D013861:Thiocyanates
D004220:Disulfides; D005353:Fibronectins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D013861:Thiocyanates; D005780:Gelatin; D003094:Collagen; D010434:Pepsin A; D016650:Fluorescein-5-isothiocyanate