Figure 5.

Effect of nucleotide concentration on release of bound GTP[γS] from ARF3 with or without cytohesin-1. ARF3 (1.5 μg, 75 pmol) was incubated with 4 μM [³⁵S]GTP[γS], as described in Fig. 4, and cooled. Samples (20 μl) were then incubated with (▪, •) or without (□, ○) cytohesin-1 (0.5 μg), and the indicated concentration of GTP[γS] (○, •) or GDP (□, ▪) for 40 min at 36.5°C before assay of protein-bound [³⁵S]GTP[γS]. One hundred percent binding (3.1–3.2 pmol) was the mean of values for ARF3 ± cytohesin-1 and unlabeled nucleotide incubated at 0°C for 60 min. Control values (0.06–0.08 pmol bound in the absence of ARF3) have been subtracted.