Evidence for preferential proteolytic cleavage of one of the two fibronectin subunits and for immunological localization of a site distinguishing them
Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin‐binding and heparin‐binding fractions were analyzed in NaDodSO4‐polyacrylamide gel electrophoresis followed by immunoblotting with a defined monocl...
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Veröffentlicht in: | European journal of biochemistry 1983-09, Vol.135 (2), p.203-207 |
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Sprache: | eng |
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Zusammenfassung: | Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin‐binding and heparin‐binding fractions were analyzed in NaDodSO4‐polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti‐fibronectin antibody. In early cathepsin G digests, several gelatin‐binding fragments were detected: a few large (Mr 150000) polypeptides and fragments of Mr= 85000, 72000, 64000 and 40000. The 85000‐Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72000‐Mr and 40000‐Mr fragments did not. Therefore the 64000‐Mr fragments are likely to be derived from the 85000‐Mr fragments. Three large fragments that bound to heparin, but not to gelatin were detected: Mr= 145000, 135000 and 120000. Of these only the 135000‐Mr peptide reacted with the antibody. When fibronectin was digested with thrombin, polypeptides of Mr= 180000–200000 and a 30000‐Mr NH2‐terminal fragment were produced. Cathepsin G added to this mixture further cleaved the fragments to a digestion pattern resembling that obtained from intact fibronectin except that the 85000‐Mr and 64000‐Mr fragments appeared as single bands and the amount of the 72000‐Mr fragment was reduced. The results suggest that thrombin cleaves the 30000‐Mr fragment preferentially from the NH2‐terminal end of one of the two subunits of fibronectin and that the 85000‐Mr, 72000‐Mr and 64000‐Mr fragments obtained by the additional cathepsin G digestion were derived from the other chain. The results are consistent with the model that the antigenic determinant resides 72000–85000 Da from the NH2‐terminus and is cleaved by cathepsin G alternatively at one of its sides. Thus, the components of the 85000‐Mr and 64000‐Mr doublets are derived from different subunits and the region located by the antibody may be responsible for the difference in their migration in the polyacrylamide gel. |
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ISSN: | 0014-2956 1432-1033 |
DOI: | 10.1111/j.1432-1033.1983.tb07638.x |