Location of a gelatin-binding region of human plasma fibronectin.

Plasma fibronectin, which is also known as cold-insoluble globulin, consists of two polypeptide chains of approximately 250,000 daltons joined by disulfide bonds located near one end of the molecule. Proteolytic digestion of human plasma fibronectin and NHz-terminal sequence analysis of some of the fragments produced were used to locate the gelatin-binding region of fibronectin within the intact molecule. Fibronectin was cleaved with cathepsin Dl and a 72,000-dalton gelatinbinding fragment was isolated. This fragment could be cleaved further with thrombin to produce a 43,000-dalton fragment which retained the ability to bind to gelatin, and a 29,000-dalton disulfide-enriched fragment which did not. All three fragments appeared to consist of single polypeptide chains. NHI-terminal analysis of the 72,000-dalton and 29,000-dalton fragments by the 5dimethylaminonaphthalene-1-sulfonyl (dansyl) chloride method before and after treatment with L-pyroglutamyl-peptide hydrolase indicated that they shared the same NHz-terminal sequence, pyroglutamic acid (tG1u)-Ala-, as intact fibronectin. Automated amino acid sequence analysis of the 72,000-dalton piece following L-pyroglutamyl-peptide hydrolase digestion confirmed that the first 6 residues of this fragment, cGlu-Ala-Glx-Glx-Met-Val-, were identical with those of intact fibronectin. The 43,000-dalton gelatin-binding fragment contained an NHz-terminal alanine residue as monitored by dansylation. These results indicate that the 29,000-dalton fragment is located at the NHz terminus of fibronectin and that a gelatin-binding site lies within an adjacent 43,000-dalton region.