hIAN1 is a GDP/GTP binding protein.

(A) Sequence of nucleotide-binding motifs G1, G3, and G4 from Ras, Gα, hIAN1, and mIAN-1. The amino acids written in bold letters have been shown to be conserved in all GTP-binding proteins; positions 12, 59, and 61 of Ras are the amino acids involved in GDP/GTP binding and GTPase activities. (B) Affinity purification of GST-hIAN1. After purification on glutathione-sepharose beads, material from a noninduced (lane 1) and induced culture of recombinant bacteria (lane 2) was loaded on a 10% SDS-PAGE, followed by staining with Coomassie blue. (C) Kinetics of GDP binding on hIAN1 protein in the presence of 10 mM MgCl₂. First, 60 nM GST-hIAN1 was incubated with 5 μM [³H]GDP in 50 mM Tris (pH 7.5), 1 mM DTT, 100 μg/mL BSA, and 10 mM MgCl₂ at 25°C. The binding of [³H]GDP was measured by filtration as described in “Materials and methods.” All measures were performed in triplicate. (D) Specificity of nucleotide binding to GST-hIAN1. First, 60 nM of GST-hIAN1 was incubated at 25°C for 120 minutes with 5 μM [³H]GDP in 50 mM Tris (pH 7.5), 1 mM DTT, 100 μg/mL BSA, and 10 mM MgCl₂ in the presence of the indicated concentrations of various nucleotides. The amount of [³H]GDP-bound protein was measured as described in panel B. □, GppNHp; ●, GDP; ×, GMP; −, ATP; ⧫, TTP; ▵, CTP.