Figure 2. Effect of nucleotides on the reactivation of wild-type and S120G mutant NDP kinase A.

(A) Kinetics of reactivation. Urea-unfolded wild-type (squares) and S120G mutant (circles) NDP kinase A was diluted 20-fold in buffer in the absence (open symbols) or presence of 0.1 mM MgATP (closed symbols). The effects of 1 mM ADP (▲) and of 1 mM p[NH]ppA (♦) on the S120G mutant is also shown. The protein was used at a concentration of 5 μg/ml. (B) Extent of S120G mutant reactivation after 2 h in the presence of ATP (circles), ATP+100 μM ADP (squares), ATP+100 μM p[NH]ppA (▲) with 5 mM MgCl₂ (closed symbols) and 1 mM EDTA (open symbols). The reaction was followed in the presence of the indicated concentration of ATP. (C) Reactivation of urea-unfolded S120G mutant diluted in buffer containing MgCl₂. ATP, at a concentration of 100 μM, was present at time zero (●) or was added after 30 min (○). Triangles represent reactivation in the absence of ATP.