Mechanism of formation of disulfide-bonded multimers of plasma fibronectin in cell layers of cultured human fibroblasts.

The free sulfhydryl groups of purified plasma fibronectin were blocked by alkylation with iodoacetamide in 3 M guanidine hydrochloride at pH 7.0 (to form fCam-fibronectin). 125I-fCam-fibronectin bound to cell layers of cultured human fibroblasts with kinetics similar to binding of 125I-labeled underivatized fibronectin. Once bound to the cell surface, 125I-fCam-fibronectin, like 125I-fibronectin, became incorporated into a detergent-insoluble extracellular matrix. Matrix-bound 125I-fCam-fibronectin, like matrix-bound 125I-fibronectin, was present as disulfide-bonded multimers as well as disulfide-bonded dimers. These results indicate that the mechanism of formation of disulfide-bonded fibronectin multimers does not involve oxidation of free sulfhydryls. Catheptic D digests of matrix-bound fibronectin were analyzed by gel electrophoresis without and with reduction. In non-reduced digests, the disulfide-rich 70-kDa amino-terminal fragment was found in a large (greater than 300 kDa) complex. Thus, fibronectin multimer formation probably occurs by disulfide exchange in the amino-terminal portion of the molecule.