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. 2001 Dec 18;98(26):14871–14876. doi: 10.1073/pnas.261419298

Figure 3.

Figure 3

Actin nucleation by Arp2/3 complex requires hydrolyzable ATP. (A) As a control, we tested elongation of 2 μM MgATP-actin from F-actin seeds in the presence of 100 μM ATP, ADP, and AMP-PNP. Kinetics of polymerization are independent of solution nucleotide within the first 100 s of starting the reaction but diverge soon after this, because of the exchange of nucleotide with solution. We therefore limited our observations to the first 100 s when using ATP-actin. (B) The Arp2/3 complex induces polymerization of MgATP-actin in the presence of ATP or ADP-BeF3 but not AMP-PNP or ADP. We used the fact that nucleotide in solution exchanges onto Arp2/3 complex within seconds (Fig. 1E) but takes much longer to exchange onto actin monomers (38) to load one type of nucleotide onto the Arp2/3 complex, but keep ATP bound to actin. We mixed 2 μM MgATP-actin with a solution of 10 nM Arp2/3 complex and 1.2 μM N-WASP WA (3-fold above saturating concentration) with 100 μM ATP, ADP, ADP + 2 mM BeF3, or AMP-PNP.