Abstract
Antisera against a Mr = 60,000 peptide containing the gelatin-binding domain of human plasma fibronectin (McDonald, J. A., and Kelley, D. G. (1980) J. Biol. Chem. 255, 8848-8858) bound the Mr = 60,000 peptide and intact fibronectin but not three other fragments released by leukocyte elastase proteinolysis (the Mr = 25,000 amino-terminal sequence, Mr = 140,000 sequence containing cell adhesive activity, and a Mr = 31,000 fragment). Affinity-purified Fab' blocked Mr = 60,000 peptide binding to gelatin and inhibited plasma and cellular fibronectin gelatin binding without affecting fibronectin-mediated cell spreading. In contrast, antifibronectin Fab' absorbed with the gelatin-binding fragment completely blocked fibronectin-mediated cell spreading. These data indicate that the gelatin-binding domain of fibronectin is immunogenic, and antisera against this domain recognize cellular fibronectin gelatin-binding sites. Inhibition of gelatin binding but not cell spreading by anti-gelatin binding domain Fab' confirms the hypothesis that fibronectin has separate sites mediating these activities. Selective inhibition of fibronectin-collagen binding by domain-specific antisera may help elucidate the role of fibronectin in organization of the extracellular matrix.
Highlights
From the Pulmonary Division, Department of Medicine, Washington University School of Medicine at The JewishHospital of St
We have utilized proteolytic cleavage to isolate the gelatin-binding domain of fibronectin, and in thisreport we describe the effects of antiserum specific for the gelatin-binding domain of human
A, fibronectingelatin substrate; H,gelatin alone; C, tibronectinincubated with 100 pg/ml of anti-60 k Fab’ before addition to gelatin plate; D,identical with C, except fibronectin incubated with preimmune Fab‘; IC, gelatin plate incubated with 100 p g /
Summary
8,000 (antiserum dilution giving 50% specific binding porfobe) added to the gelatin-coated substrate prior to the addition of of anti-60 k antiserum developed by 2-3 months after initial cells, demonstrating selective inhibition of fibronectin-gelatin injection. To phoresis of affinity-purified and preimmune IgGgaveone verify these results, similar experiments were carried out on band of Mr = 150,000 (unreduced) and bands at M , = 50,000 substrates coated with fibronectin alone. A, fibronectingelatin substrate; H ,gelatin alone; C, tibronectinincubated with 100 pg/ml of anti-60 k Fab’ before addition to gelatin plate; D,identical with C, except fibronectin incubated with preimmune Fab‘; IC, gelatin plate incubated with 100 p g /. Note the marked ‘, inhibition of spreading when fibronectin is incubated with anti-60 k Fab’before addition to gelatin substrate and the lack.
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