Selective Pharmacological Targeting of a DEAD Box RNA Helicase
Figure 3
Characterization of eIF4A hippuristanol-resistant mutants.
(A) Consequence of mutations in the eIF4AI hippuristanol binding site on ATP hydrolysis. ATP hydrolysis was monitored using 1 µg His6-eIF4AI or His6-eIF4AIIG/T in the presence or absence of 10 µM hippuristanol. Each value represents the average of three measurements with the standard deviation presented. (B) Relative ATPase activity of eIF4A mutants in the presence of hippuristanol. The percent ATP hydrolysis was determined in the presence of hippuristanol and set relative to the values obtained in the presence of control reactions containing vehicle (DMSO). The results represent the average of 3–7 experiments with error bars signifying the standard deviation. (C) Altered hippuristanol sensitivity of eIF4AIII. ATPase assays were performed with 0.5 µg recombinant protein with 0.1 µCi γ-32P-ATP (10 Ci/mmol). Following analysis by TLC and quantitation using a Fuji BAS 2000 phosphoimager, the percent hydrolysis was determined and set relative to the DMSO vehicle control reactions. Each value represents the average of three measurements with the standard deviation shown.