Isolation and characterization of a heparin-binding domain of cellular fibronectin.

A protease-resistant functional polypeptide domain of fibronectin that contains a heparin-binding site has been purified from chick cellular fibronectin following pronase digestion, heparin-Sepharose affinity chromatography, and molecular sieve chromatography on Sephacryl S-200. This polypeptide migrates as a single band of apparent Mr = 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reduction by dithiothreitol. It has a Stokes radius of 35 to 37 A and a calculated frictional ratio of 1.4 to 1.5. Its amino acid composition is unusual in that cysteine and methionine constitute only 1 and 2 mol/mol of the polypeptide, respectively. The NH2-terminal amino acid sequence is Glu-Asp-Asp. In contrast to intact fibronectin, the heparin-binding polypeptide contains virtually no neutral sugars. This 50,000-dalton domain is inactive in four bioassays for intact fibronectin, including hemagglutination, spreading of cells on tissue culture plastic, restoration of alignment of tumor cells, and attachment of cells to collagen. Moreover, excess amounts of this polypeptide do not inhibit the activity of intact fibronectin in all of these assays; these results suggest that the heparin-binding domain does not contain a cell surface-binding site. This domain of fibronectin appears to be a unique region free of carbohydrate that is a globular, protease-resistant functional site.