Ca2(+)-sensitive binding of thrombospondin to U937 cells is due to the formation of calcium precipitate in the binding medium.
Thrombospondin (TSP) binds to U937 monocytic cells in a Ca2(+)-enhancible and EDTA-inhibitable manner (Silverstein, R. L., and R. L. Nachman. 1987. J. Clin. Invest. 79:867-874; Silverstein, R. L., A. S. Asch, and R. L. Nachman. 1989. J. Clin. Invest. 84:546-552). We reproduced the results when RPMI cell culture medium, but not when HBSS was used as binding medium. Addition of 1 mM Ca2+ to RPMI medium increased the binding of TSP to suspended U937 cells more than eightfold; the increase was blocked by EDTA but not by heparin. Further studies showed that addition of 1 mM Ca2+ to RPMI medium resulted in an insoluble precipitate, which did not form when EDTA was present or when 1 mM extra Ca2+ was added to HBSS. TSP bound to the precipitate in a saturable and specific manner. The precipitate enhanced binding of TSP to MG63 osteosarcoma cells in a monolayer binding assay. Enhancement of binding in the monolayer assay was observed for fibronectin and vitronectin as well. Our data indicate that there is not a specific Ca2(+)-dependent TSP receptor on U937 cell surface. Instead, the extra binding enhanced by Ca2+ is due to the formation of insoluble salts in the medium.
Sun
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Department of Medicine, University of Wisconsin, Madison 53706.
Mosher
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D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D011232:Chemical Precipitation; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007938:Leukemia / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D019699:Thrombospondins; D014407:Tumor Cells, Cultured
D010980:Platelet Membrane Glycoproteins; D019699:Thrombospondins; D004492:Edetic Acid; D002118:Calcium