Fig. 2. T27N mutation decreases the affinity of Arf6 for both GDP and GTPS and destabilizes the protein. T44N mutation affects the binding of GTPS but not of GDP. (A,B) Arf6 wt(), Arf6-T27N() or Arf6-T44N() were loaded for 40 minutes at 37°C with [³H]-GDP (A) or [³⁵S]-GTPS (B) in the presence of 1 µM free Mg²⁺. Then, the free Mg²⁺ concentration was raised to 1 mM and [³H]-GDP and [³⁵S]-GTPS dissociations were initiated by adding 1 mM unlabeled GDP or GTPS, respectively. The curves are best fits assuming first-order kinetics for nucleotide dissociation; nucleotide dissociation rates are shown on the left. (C) Arf6 T27N () or T44N () were loaded at 37°C with [³H]-GDP in the presence of 1 µM free Mg²⁺. Then, the free Mg²⁺ concentration was raised to 1 mM and, without isotopic dilution, the radioactivity bound to the proteins was measured over time. (D) Arf6-T27N or Arf6-T44N (5 µM) was incubated in the presence of 50 µM GDP and 1 mM free Mg²⁺ for 2 hours at 37°C. After ultracentrifugation, pellet (P) and supernatant (S) were analysed by SDS-PAGE and Coomassie Blue staining. The distribution (as percentages) of Arf between pellet and supernatant is indicated.