Purification and Characterization of Proteoglycan from Bovine Aortic Endothelial Cells Conditioned Media, and Its Interaction with Basic Fibroblast Growth Factor (bFGF)
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A protocol to investigate the cell free binding of bFGF with purified BAE PG was established using the BioRad Bio-Dot apparatus - the cationic filtration assay (CAFAS). Using a simple monovalent binding model, we obtained values for the equilibrium dissociation constant, KD, of (1.6 Â± 0.8) x 10 -10 M; the dissociation rate constant, kr, of 0.01 min -1; the association rate constant, kf, of 6.2 x 10 7 M -1 min -1 and the total binding sites of the proteoglycan, RT, of 0.1~0.2 (# of site)/(molecule of PG). The comparison of experimental data with model predictions indicates that when the number of binding sites provided by the PG is similar or greater than that of bFGF, the monovalent binding model is valid. When the number of binding sites is less than that of bFGF, one possibility is that the binding might not be the described simple monovalent reaction, and bFGF might bind to the PG as dimers or oligomers. In addition, a model is proposed for BAE PG, in which 5 ~ 10 BAE PG molecules form a high affinity binding site for bFGF. Experimentally we find that exogenous heparan sulfate competes with BAE PG for binding with bFGF, while chondroitin sulfate seems to facilitate the binding. This result may be a useful consideration when we want to design possible pharmaceutical compounds.
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