Regular fragmentation of hydrogen peroxide-treated fibronectin.
In the presence of low concentrations (less than 0.5 mM) of hydrogen peroxide Mr 350,000 and 170,000 fragments were generated from plasma and fibroblast medium fibronectins (Fns). No other major fragments were detected when H2O2 concentration was raised or the incubation time prolonged. A 200-300-fold concentration of H2O2 was needed for a complete degradation of the protein. The degradation was inhibited or completely prevented by deferoxamine, diethylene-triaminepentaacetic acid, and thiourea or by Chelex-pretreatment of the Fn solution suggesting a Fenton-type reaction to produce .OH radicals from H2O2. In immunoblotting the Mr 170,000 fragment reacted with monoclonal antibodies against the NH2 terminus and mid-molecule but not with those against the cell-binding site and the COOH terminus of Fn. Reduction of the Mr 350,000 fragment produced alpha- and beta-monomers of Fn as well as Mr 95,000 and 85,000 fragments which reacted with monoclonal antibodies against the cell-binding site and the COOH terminus of Fn. These results suggest that the Mr 170,000 fragment is derived from the NH2-terminal part of both subunits of Fn. The rest of the subunits, the Mr 95,000 (from alpha-chain) and Mr 85,000 (from beta-chain), thus remain disulfide-bonded to an intact Fn subunit to form the nonreduced Mr 350,000 polypeptide. The results show that oxygen radical action may generate defined and reproducible fragments from Fn. The high susceptibility of Fn to the radical induced degradation makes it plausible to occur also in vivo.
Vartio
T
T
Department of Pathology, University of Helsinki, Finland.
D001665:Binding Sites; D003676:Deferoxamine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000378:metabolism*; D005609:Free Radicals; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006878:Hydroxides / Q000378:metabolism; D017665:Hydroxyl Radical; D015151:Immunoblotting; D046911:Macromolecular Substances; D008970:Molecular Weight; D004369:Pentetic Acid / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism*; D011137:Polystyrenes / Q000494:pharmacology; D011145:Polyvinyls / Q000494:pharmacology; D013890:Thiourea / Q000494:pharmacology
D005353:Fibronectins; D005609:Free Radicals; D006878:Hydroxides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011137:Polystyrenes; D011145:Polyvinyls; D017665:Hydroxyl Radical; D004369:Pentetic Acid; C006960:chelex; D006861:Hydrogen Peroxide; D013890:Thiourea; D003676:Deferoxamine