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. Author manuscript; available in PMC: 2011 Dec 22.
Published in final edited form as: Biochemistry. 2009 Mar 3;48(8):1754–1762. doi: 10.1021/bi801291q

Figure 4. NSC 622124 is a Nanomolar Inhibitor of HsEg5 MT-Stimulated ATPase Activity and Competes with MTs but not ATP for Association with HsEg5.

Figure 4

(A) MT-stimulated ATPase activity as a function of NSC 622124 concentration. Reactions containing 100 nM HsEg5, paclitaxel-stabilized MTs (0.5 µM tubulin), and 1 mM MgATP were prepared with a range of NSC 622124 concentrations (0, 0.002, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 25, 50, and 100 µM) and ATPase rates were determined with a malachite green assay. Each point represents the mean ± SEM of 2–16 reactions. All data were used to fit the curve although only data from 0–25 µM NSC 622124 are shown. (B) MT-stimulated ATPase rates of 100 nM HsEg5 were measured in the presence of 2 mM MgATP and 0, 50 or 100 nM NSC 622124 at several tubulin concentrations (0.17, 0.27, 0.37, 0.64, and 1.08 µM), and results were graphed as a Lineweaver-Burk plot. Each data point represents the mean of 4–12 reactions. SEM values averaged 4% of the means (range = 2% to 8%). (C) MT-stimulated ATPase rates of 100 nM HsEg5 were measured in the presence of MTs (0.75 µM tubulin) and 0, 50 or 150 nM NSC 622124 over a range of MgATP concentrations (62.5, 125, 250, and 500 µM), and results were graphed as a Lineweaver-Burk plot. Each data point represents the mean of 4–8 reactions. SEM values averaged 4% of the means (range = 1% to 7%).