Interaction between cell-binding domain and extracellular matrix-binding domain of fibronectin determined by fluorescence depolarization.
Interaction of domains in fibronectin was observed by photometry of fluorescence polarization of three kinds of dye; [N-(1-anilinonaphthyl-4)]maleimide (ANM tau = 5 ns), [N-(3-fluoranthyl)]maleimide (FAM tau = 20 ns), and [N-(3-pyrene)]maleimide (PRM tau = 100 ns). Each dye was labeled at a free sulfhydryl group in the cell-binding domain. Neither fluorescence of ANM with short fluorescent lifetime, FAM with long lifetime, nor PRM with longer fluorescent lifetime on fibronectin depolarized as much as the free dye. It was found that each dye was firmly fixed in the cell-binding domain. When heparin or gelatin was added in the solution of PRM-fibronectin complex, the fluorescence polarization tended to increase principally by combining heparin or gelatin to fibronectin. It was found that the rotation of whole or partial fibronectin containing the cell-binding domain through fluorescent lifetime of 100 ns was suppressed by combining of heparin or gelatin to fibronectin. When heparin or gelatin was added in the solution of ANM- or FAM-fibronectin complex, on the contrary, the fluorescence polarization tended to decrease, that is, slightly depolarize through the fluorescent lifetime of 5 or 20 ns, respectively. It was found that the rotation of the cell-binding domain, or of part of the fibronectin molecule containing the domain, was slightly promoted by combining heparin or gelatin to its domain. These results indicate that an interaction of the heparin- or gelatin-binding domain with the cell-binding domain was induced by the combining of heparin or gelatin to the respective domains.
Miyamoto
Y
Y
Institute of Biological Sciences, University of Tsukuba, Ibariki, Japan.
Yokoya
A
A
Ishizaka
S
S
D000818:Animals; D005109:Extracellular Matrix / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D005780:Gelatin / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D007202:Indicators and Reagents; D008301:Maleimides / Q000378:metabolism; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D013552:Swine; D014357:Trypsin
D005353:Fibronectins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008301:Maleimides; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C055605:N-(3-fluoranthyl)maleimide; C010854:N-(1-anilinonaphthalyl)-4-maleimide; D005780:Gelatin; D006493:Heparin; C003944:N-(3-pyrene)maleimide; D014357:Trypsin