Determination of the hIAN1 affinity for GDP and GppNHp.

First, 60 nM GST-hIAN1 protein was incubated for 120 minutes at 25°C with various concentrations of GDP (0.175, 0.3, 0.7, 1.5, 3, 5, and 15 μM) or GppNHp (2, 4, 8, 10, 16, 30, 60, 80, 100, and 120 μM); then, the amounts of bound nucleotide were determined by filter-binding assays. All measures were performed in triplicate. Panels A and C show the concentration-dependent binding of GDP (panel A) and GppNHp (panel C) to GST-hIAN1. Scatchard representations are shown in panel B (GDP) and panel D (GppNHp). r indicates bound nucleotide/total protein (pmoles); F is the concentration of free nucleotide (micromolar).