FIGURE 4.
Effect of (−)-maackiain on Hsp90 enzymatic activity. A, geldanamycin competition assay. Recombinant Hsp90α (0.7 μg) and FITC-labeled geldanamycin (5 nm) were incubated with or without various concentrations of (−)-maackiain (●) or 17-AAG (■) for 2 h at 25 °C. Then fluorescence was measured at λex = 485 nm and λem = 530 nm using a microtiter plate reader. B, Hsp90 ATPase assay. Recombinant Hsp90α (2 ng) and 1 mm ATP were incubated with or without various concentrations of (−)-maackiain (●) or 17-AAG (■) for 16 h at 37 °C. The reaction was stopped by the addition of malachite green reagent. 15 min after the addition of sodium citrate, the absorbance at 620 nm was measured using a microtiter plate reader.