title: Isolation of an amino-terminal fibronectin-binding protein on human U937 cells and rat peritoneal macrophages.
abstract: Several cell-mediated activities for the amino terminus of fibronectin have been documented. In the present study we describe a macrophage surface protein with binding activity directed to the amino terminus of the fibronectin molecule. The binding of a 29-kDa amino-terminal fibronectin fragment to macrophages reached steady state by 30 min and was half-maximal at approximately 2 x 10(-8) M. This binding was specifically inhibited by excess unlabeled 29-kDa fragment or intact fibronectin but not by a 180-kDa fibronectin fragment which lacks the amino terminus. Competitive binding studies of the 70-kDa amino-terminal fibronectin fragment to macrophages revealed a single binding site with KD = 7.14 x 10(-8) M and approximately 8 x 10(4) binding sites/cell. Radiolabeled surface proteins extracted from rat peritoneal macrophages and from the human U937 cell line were applied to an affinity column comprised of the 70-kDa amino-terminal fragment of fibronectin coupled to a solid support. A single trypsin-sensitive radiolabeled protein of 67 kDa, from either cell type, was eluted from this column with urea. This protein showed no immunologic identity with fibronectin, fibrin(ogen), or albumin. The 67-kDa protein exhibited identical apparent molecular weight under reducing and nonreducing conditions, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. We have localized the fibronectin binding activity of this protein to within the 29-kDa amino-terminal domain of fibronectin. The 67-kDa protein eluted from the 70-kDa column failed to bind to a column comprised of the 45-kDa gelatin-binding fragment of fibronectin. Additionally, the 67-kDa protein was specifically eluted from the 70-kDa column by the 29-kDa amino-terminal fragment but not by the 45-kDa gelatin-binding fragment. These data suggest that this 67-kDa protein is a macrophage cell surface binding protein for the amino terminus of fibronectin.
authors.affiliation: Department of Physiology and Cell Biology, Albany Medical College, New York 12208.
authors.forename: S D
authors.initials: SD
authors.lastname: Blystone
authors.forename: J E
authors.initials: JE
authors.lastname: Kaplan
mesh_terms: D000418:Albumins / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D008264:Macrophages / Q000737:chemistry* / Q000378:metabolism; D010529:Peritoneal Cavity / Q000166:cytology; D011233:Precipitin Tests; D051381:Rats; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000302:isolation & purification* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D000418:Albumins; D005353:Fibronectins; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic; D005780:Gelatin; D005340:Fibrinogen; D014357:Trypsin