A probability value of less than 0. 01 was considered statistically significant. Figure legends Erlotinib structure specify statistically significant differences between experimental groups at probability values of p 0. 01 and p 0. 001. Analysis was performed using WinSTAT. Results Binding of ATP to VEGF A165 In order to evaluate the binding of ATP to VEGF Inhibitors,Modulators,Libraries A165, VEGF A165 was radiolabeled by ATP and ATP. The use of ATP as well as ATP allowed to distinguish between binding of ATP to VEGF A165 and autophosphorylation. The influence of divalent cations was also tested. Signal is detected for both ATP and ATP labeled growth factor independently of the presence of Mg2. ATP appeared to be bound to growth factor by non covalent interaction via the phosphate residues of the nucleotide.

In contrast to a covalent modification, Inhibitors,Modulators,Libraries an ionic interaction can be influenced by an increase in ionic strength. Labeling of VEGF A165 with ATP and ATP, respectively, was sup pressed by 100 mM NaCl added to the reaction mixture prior to the nucleotides. Once the complex Inhibitors,Modulators,Libraries had formed, however, it proved to be fairly resistant to the salt concentration. VEGF A165 contains a heparin binding domain which is critical for its mitogenic activity and storage in the extra cellular matrix via HSPGs. Interestingly, heparin affected binding of ATP to FGF 2 due to overlapping binding sites. Competition experiments revealed that heparin also interfered with the binding of ATP to VEGF A165. 10 ug mL heparin added to the reaction mixture prior to ATP reduced radiolabeling of VEGF A165 markedly. 100 ug mL heparin inhibited ATP binding to the mitogen completely.

However, when ATP was added to the reaction mixture prior to heparin, only a slight decrease Inhibitors,Modulators,Libraries in radiolabel ing of VEGF A165 occurred. MALDI TOF MS of the VEGF A165 ATP complex MALDI TOF MS was performed employing soft condi tions as described previously. This approach was suitable for the detection of labile nucleotide protein complexes. Sample preparations using low acidic reversed phase chromatography and acid free matrix assisted in retaining Inhibitors,Modulators,Libraries the non covalent interaction. The measurements were performed using the high mass detector in order to observe the VEGF A165 dimer as the bioactive species present in vivo. The incubation of VEGF A165 with ATP caused considerable peak broadening as compared to pure VEGF A165.

The shift in mass corresponded to the addition of one molecule of ATP per molecule of growth factor and did not selleck chem Veliparib differ when Mg2 was added to the incubation mixture. The dimer was also affected by ATP, but the number of bound ATP molecules could not be clearly defined due to low signal intensity. ATP induces a conformational change of VEGF A165 Far UV CD spectroscopy was carried out in order to investigate a putative effect of ATP binding on the second ary structure of VEGF A165. Thus, the CD of VEGF A165 was measured without or with a twofold molar excess of ATP.