Formation of sodium dodecyl sulfate-stable fibronectin multimers. Failure to detect products of thiol-disulfide exchange in cyanogen bromide or limited acid digests of stabilized matrix fibronectin.
Fibronectin exists in a soluble form in body fluids and as a fibrillar component of the extracellular matrix. Matrix fibronectin associates as large complexes in SDS unless a reducing reagent is also present. This observation suggests that complex formation is due to interprotomeric disulfides that form by thiol-disulfide exchange. To localize the presumptive new disulfides, we labeled protomeric fibronectin by the chloramine-T method or with 125I-Bolton-Hunter reagent, incorporated 125I-fibronectin into the matrix of cultured fibroblasts, and subjected matrix fibronectin to acid or cyanogen bromide digestion. When cyanogen bromide digests of matrix 125I-fibronectin and protomeric 125I-fibronectin labeled with Bolton-Hunter reagent were analyzed by two-dimensional polyacrylamide gel electrophoresis in SDS, with the first dimension being nonreducing and the second reducing, we were not able to identify any fragments of matrix fibronectin that migrated as high molecular weight complexes in the first dimension. Limited acid digestion of matrix 125I-fibronectin also dissociated the majority of the high molecular weight complexes. Since we could account for all of the parts of fibronectin that contain cysteine or cystine, we conclude that matrix fibronectin is not stabilized by interprotomeric disulfides. We propose, instead, that stabilization is mediated by noncovalent protein-protein interactions that are sensitive to reduction, cyanogen bromide digestion, or limited acid digestion.
Chen
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Department of Medicine, University of Wisconsin, Madison, 53706, USA.
Mosher
D F
DF
D000143:Acids; D003488:Cyanogen Bromide; D004220:Disulfides / Q000737:chemistry; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D005353:Fibronectins / Q000737:chemistry*; D006801:Humans; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D012967:Sodium Dodecyl Sulfate
D000143:Acids; D004220:Disulfides; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012967:Sodium Dodecyl Sulfate; D003488:Cyanogen Bromide