Location of Heparin-Binding Sites of Fibronectin. Detection of a hitherto Unrecognized Transamidase Sensitive Site

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Abstract
Resolution of a cathepsin D digest of plasma fibroncectin on heparin-Sepharose yielded, in addition to non-bound and weakly retained material (Fraction I and II), various fragments which were not eluted until 0.25M NaCl (Fraction III) and 0.5M NaCl (Fraction IV) was applied. Fraction III contained predominantly a peptide of Mr [MW] 70,000 originating from the N-terminus of the fibronectin subunits and high MW precursors yielding the MW 70,000 peptide by further digestion. All those peptides were retrained by immobilized denatured collagen, type I, indicating the presence of the known gelatin-binding domain. In addition, they contained a transamidase-sensitive site as revealed from a digest of fibronectin previously labelled with [14C]putrescine by a transamidase-mediated reaction. Plasminolysis of the fragment of Mr 70,000 caused two peptides of Mr 30,000 and 40,000, only the former being retained by heparin-Sepharose. Fraction IV contained a fragment of MW 140,000 which, after reduction, dissociated into 2 peptides of MW 75,000 and 65,000. Apparently, it included the disulfide bond(s) connecting the 2 fibronectin subunits close to their C-terminal ends. Partial digestion of the 2 electrophoretically separated peptide chains with protease of Staphylococcus aureus V8 yielded for each chain a number of peptides with equal electrophoretic migration rate. In addition some peptides were different in the 2 digests. The results were consistent with an identical or homologous structure of the 2 peptide chains with an additional sequence in the longer chain. The latter (Mr 75,000) uniquely contained a transamidase susceptible site as demonstrated by processing of [14C]putrescine-labeled fibronectin.

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