学術雑誌論文

学術雑誌論文

text

Journal of Biological Chemistry. 1985, 260(8), p. 5105-5114

Differences in domain structure between human fibronectins isolated from plasma and from culture supernatants of normal and transformed fibroblasts : Studies with domain-specific antibodies

American Society for Biochemistry and Molecular Biology

英語

text

1985-04

http://www.jbc.org/content/260/8/5105

This research was originally published in the Journal of Biological Chemistry. K Sekiguchi, A Siri, L Zardi and S Hakomori. Differences in domain structure between human fibronectins isolated from plasma and from culture supernatants of normal and transformed fibroblasts : Studies with domain-specific antibodies. J. Biol. Chem. 1985; 260: 5105-5114 © the American Society for Biochemistry and Molecular Biology

The domain structure of human fibronectins isolated from plasma and from the conditioned medium of normal and transformed fibroblasts was analyzed by limited proteolysis and S-cyanylation followed by immunostaining of released fragments with five kinds of antibodies, each specific for one functional domain. The results indicate that all three human fibronectins are composed of the same set of functional domains aligned in the same topological order. However, the following clear differences were found in specific fragments released from plasma fibronectin (pFN) and those released from fibronectin of normal (N-cFN) and transformed fibroblasts (T-cFN). Two fragments (Mr = 70,000 and 60,000) were released from the COOH-terminal region of pFN by cathepsin D. These fragments represent the COOH-terminal heparin-binding (Hep-2) and fibrin-binding (Fib-2) domains. The corresponding fragments released from both N-cFN and T-cFN by cathepsin D had much larger molecular weights (Mr = 100,000 and 83,000-74,000) than those from pFN. The fragments from the Fib-2 domain alone, however, did not show any difference among all three FNs. The internal region, from the gelatin-binding (Gel) domain through the Hep-2 domain, of N-cFN and T-cFN was released as a Mr = 210,000 fragment upon mild trypsin digestion. The corresponding fragment from pFN was released as a Mr = 185,000 fragment. The COOH-terminal half, including the Hep-2 domain, of both N-cFN and T-cFN was released by S-cyanylation as Mr = 160,000-145,000 fragments, which are 25,000-20,000 larger than the corresponding fragments of pFN. These results clearly indicate that the Hep-2 domain of N-cFN and T-cFN is 30,000-20,000 daltons larger than the same domain of pFN. Although various fragments released from N-cFN and T-cFN showed a similar pattern, there were minor differences. Thermolysin fragments derived from the Hep-2 domain of N-cFN were clearly distinguishable from those from T-cFN. Three groups of fragments with Mr = 40,000, 35,000-32,000, and 30,000 were released from N-cFN, while only the 35,000-32,000 fragment was released from T-cFN. The Mr = 44,000/60,000 thermolysin fragments representing the Gel domain and the Mr = 210,000/165,000 tryptic fragments representing the internal domains of T-cFN were slightly, but consistently, larger than those of N-cFN. A panel of domain-specific antibodies combined with well-characterized proteolytic and chemical fragmentation provides a highly sensitive and specific procedure to study the domain structure of various types of fibronectins. This procedure requires only a small quantity of protein, without prior purification, and is applicable to both soluble and insoluble fibronectins.

71445

Differences in domain structure between human fibronectins isolated from plasma and from culture supernatants of normal and transformed fibroblasts : Studies with domain-specific antibodies

American Society for Biochemistry and Molecular Biology

英語

1985-04

Journal of Biological Chemistry

260

8

5105

5114

https://hdl.handle.net/11094/71445

http://www.jbc.org/content/260/8/5105