Cell-free formation of disulfide-bonded multimer from isolated plasma fibronectin in the presence of a low concentration of SH reagent under a physiological condition.
Purified plasma fibronectin in Tris-buffered saline aggregated on incubation at 37 degrees C in the presence of dithiothreitol without the presence of cells. On SDS polyacrylamide gel electrophoresis without reduction, dimeric fibronectin showed a 460 kDa band, while the protein band of aggregated fibronectin remained at the top of the running gel. The aggregate was a disulfide-bonded multimer, since both the dimeric and the multimeric fibronectins migrated as 230 kDa polypeptides after reduction. The multimer formation required SH reagent and proceeded faster with higher SH concentration, suggesting the occurrence of a disulfide exchange reaction during the aggregation. Since dimeric fibronectin with carboxymethylated sulfhydryl groups also formed multimers under the same condition, the free sulfhydryl groups of dimeric fibronectin may not be involved in the multimer formation, suggesting involvement of disulfide exchange from intramolecular bonds to intermolecular bonds. The multimerization was not influenced by Na+, Ca2+, or EDTA, while urea-treated fibronectin required a higher concentration of dithiothreitol for multimer formation. Fibronectin partially degraded by m-calpain did not form multimers. The multimeric fibronectin retained heparin-binding and cell attachment activities, but had lost gelatin-binding activity. Involvement of the terminal regions containing type I and type II repeats was suggested in the interaction of pFN leading to the multimerization.
Sakai
K
K
Nippi Research Institute of Biomatrix, Tokyo, Japan.
Fujii
T
T
Hayashi
T
T
D000818:Animals; D002154:Calpain / Q000378:metabolism; D002417:Cattle; D002448:Cell Adhesion; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D005354:Fibrosarcoma / Q000378:metabolism; D008970:Molecular Weight; D011108:Polymers; D011485:Protein Binding; D014407:Tumor Cells, Cultured; D014508:Urea / Q000378:metabolism
D004220:Disulfides; D005353:Fibronectins; D011108:Polymers; D014508:Urea; D002154:Calpain; D004229:Dithiothreitol