Gp is a major GTP-binding protein of human placenta and platelets [Evans, T., Brown, M. L., Fraser, E. D., & Northup, J. K. (1986) J. Biol. Chem. 261, 7052-7059], High-affinity guanine nucleotide binding is associated with a polypeptide migrating identically with H-ras on SDS-PAGE. We have characterized the interactions of preparations of purified human placental G., with guanine nucleotides in detergent solution. Equilibrium binding studies with [35S]GTPΓS, [3H]Gpp(NH)p, and [3H]GTP identified a single class of sites with a dissociation constant of 10 ± 1, 153 ± 61, and 125 ± 77 nM for the ligands, respectively. These three ligands were mutually competitive with Ki values consistent with the Kd values from direct binding experiments. Competition for the binding of [3H]Gpp(NH)p was used to determine the specificity of the site. values determined from this assay were 14 nM for GTPΓS, 143 nM for Gpp(NH)p, 3.3 µM for GDPβS, 69 nM for GTP, and 64 nM for GDP. ATP, ADP, cAMP, cGMP, and NAD+ had no detectable affinity for this site. While the equilibrium binding data fit well to a single class of sites, association kinetics of these ligands were better fit to two rate constants. Dissociation kinetics, however, were not clearly resolved into two rates. All binding reaction rates were regulated by magnesium ion. In the absence of added magnesium and in the presence of EDTA, no association of the ligands was detected, and the dissociation of bound ligands was facilitated by addition of EDTA. Binding appeared to require only micromolar concentrations of magnesium, and at these concentrations, dissociation of bound ligand was markedly retarded. The binding of [3H]GTP to Gp in the presence of magnesium lead to hydrolysis of the γ-phosphoryl group, with GDP remaining tightly associated with the site. Incubation of Gp with GTP lead to hydrolysis of GTP and the formation of GDP-bound Gp. As opposed to untreated Gp, the GN-binding properties of GDP-Gp were found to be rate-limited by GDP dissociation. Guanine nucleotides stabilized the binding site against thermal inactivation. GTPΓS and Gpp(NH)p were most effective, and GDP was partially effective, while aluminum fluoride with or without GDP had no effect. Magnesium was required for the stabilization by guanine nucleotides. These data identify a metal-dependent GTP-specific binding site on Gp and describe a GTP-binding site distinct from that reported for other G-proteins or ras-related GTP-binding proteins. © 1990, American Chemical Society. All rights reserved.
