Table 2.
Number of nucleotides bound per Arp2/3 complex
| Exchange buffer | Stoichiometry of bound nucleotide
|
||||
|---|---|---|---|---|---|
| Measured
|
Normalized to
actin
|
||||
| Actin (n=4) | Arp2/3 | Arp2/3 +N-WASP | Arp2/3 | Arp2/3 +N-WASP | |
| No added ATP | 0.24 | 0.21 | 0.45 | 0.4 | |
| 1.4 μM ATP | 0.76 | 0.83 | 1.4 | 1.6 | |
| 10.0 μM ATP | 0.53 | 1.2 | 1.1 | 2.3 | 2.1 |
We investigated the total number of nucleotides bound to the Arp2/3 complex by using a buffer-exchange column to rapidly exchange the Arp2/3 complex into a low concentration of ATP and measure the total nucleotide present. We heat-denatured the Arp2/3 complex to release bound nucleotide into solution, removed protein by filtration through a 10-kD MWCO filter, and analyzed nucleotide content of the filtrate by HPLC, subtracting the contribution caused by buffer alone. To correct for systematic error, results were normalized to actin.