In vitro formation of disulfide-bonded fibronectin multimers.
Fibronectin purified from a plasma protein side fraction in the absence of denaturant contained 1.5 to 1.9 cryptic free sulfhydryl groups per 200- to 250-kDa subunit. Exposure of sulfhydryl groups in physiologic salt solutions required at least 1 M guanidine, and 3 M guanidine was required for optimal exposure. The sulfhydryl groups were not exposed by collagen, a fibronectin-binding collagen fragment, fibrinogen, heparin, hyaluronic acid, calcium ion, EDTA, deoxycholate, or methylamine. One- and two-dimensional gel electrophoresis indicated that a molecule of 40-60 kDa was disulfide-bonded to a minor portion of the fibronectin in whole human plasma and in preparations of purified fibronectin. In addition, traces of disulfide-bonded multimers were present in preparations of purified fibronectin. The proportion of fibronectin in disulfide-bonded multimers increased in guanidine-containing solutions. Compared to dimeric fibronectin, these multimers had limited solubility in physiologic buffers, could be readily cross-linked by Factor XIIIa, and exhibited altered tryptic susceptibility. In free sulfhydryl groups were blocked by prior alkylation with N-ethylmaleimide or iodoacetamide, fibronectin did not form disulfide-bonded multimers in guanidine-containing solutions. The patterns of altered tryptic susceptibility and cyanide cleavage suggested that multimer formation is mediated by both sulfhydryls of fibronectin. The transition from dimeric to multimeric fibronectin can serve as a model for the formation of disulfide-bonded fibronectin multimers in the extracellular matrix.
D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005176:Factor XIII / Q000494:pharmacology; D005353:Fibronectins / Q000097:blood*; D005455:Fluorescent Antibody Technique; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D046911:Macromolecular Substances; D011190:Potassium Cyanide / Q000494:pharmacology; D012995:Solubility; D013438:Sulfhydryl Compounds; D011503:Transglutaminases; D014357:Trypsin
D004220:Disulfides; D005353:Fibronectins; D006146:Guanidines; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D005176:Factor XIII; D011503:Transglutaminases; D014357:Trypsin; D019791:Guanidine; D011190:Potassium Cyanide