FIGURE 7. The actin cross-linking reaction requires ATP as a cofactor.
Purified LFNACD was incubated with (1) 30 μM Mg-ADP-actin or (2) 30 μM Mg-ATP-actin at a molar ratio of 1:500 in 2mM MgCl2, and actin cross-linking was assayed after 60 min. 10 μM actin, further purified by gel filtration, was added to LFNACD and Mg2+, supplemented with either (3) 0.5 mM AMP-PNP or (4) 0.5 mMATP, and the reaction was terminated after 12 min. All samples were analyzed by SDS-PAGE, and cross-linked actin species were detected by Coomassie Brilliant Blue R-250.