INTERACTION OF NUCLEOTIDES WITH ACIDIC FIBROBLAST GROWTH-FACTOR (FGF-1)

A wide variety of nucleotides are shown to bind to acidic fibroblast g rowth factor (aFGF) as demonstrated by their ability to (1) inhibit th e heat-induced aggregation of the protein, (2) enhance the thermal sta bility of aFGF as monitored by both intrinsic fluorescence and CD, (3) interact with fluorescent nucleotides and displace a bound polysulfat ed naphthylurea compound, suramin, (4) reduce the size of heparin-aFGF complexes, and (5) protect a reactive aFGF thiol group. The binding o f mononucleotides, diadenosine compounds (Ap(n)A), and inorganic polyp hosphates to aFGF is enhanced as the degree of phosphorylation of thes e anions is increased with the presence of the base reducing the appar ent binding affinity. The nature of the base appears to have much less effect. Photoactivatable nucleotides (8N(3)-ATP, 2N(3)-ATP, 8N(3)-GTP , and 8N(3)-Ap(4)A) were employed to covalently label the aFGF nucleot ide binding site. In general, K-d's in the low micromolar range are ob served. Protection against 90% displacement is observed at several hun dred micromolar nucleotide concentration. Using 8N(3)-ATP as a prototy pic reagent, photolabeled aFGF was proteolyzed with trypsin and chymot rypsin and labeled peptides were isolated and sequenced resulting in t he identification of 10 possible labeled amino acids (Y8, G20, H21, T6 1, K112, K113, S116, R119, R122, H124). On the basis of the crystal st ructure of bovine aFGF, eight of the prospective labeled sites appear to be dispersed around the perimeter of the growth factor's presumptiv e polyanion binding site. One residue (T61) is more distally located b ut still proximate to several positively charged residues, and another (Y8) is not locatable in crystal structures. Using heparin affinity c hromatography, at least three distinct photolabeled aFGF species were resolved. These labeled complexes display diminished affinity for hepa rin and a reduced ability to stimulate mitogenesis even in the presenc e of polyanions such as heparin. In conclusion, nucleotides bind appar ently nonspecifically to the polyanion binding site of aFGF but nevert heless are capable of modulating the protein's activity. Evidence for the presence of a second or more extended polyanion binding site and t he potential biological significance of these results in terms of pote ntial natural ligands of aFGF are also discussed but not resolved.