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. 2016 Apr 28;291(26):13571–13579. doi: 10.1074/jbc.M116.724328

FIGURE 5.

FIGURE 5.

Effects of R1490A/K1491A mutations on the regulation of Myo5a-FL and the activation by Mlph-GTBDP. A, the ATPase activities of Myo5a-FL under EGTA and pCa4 conditions. The ATPase activity of Myo5a-FL was measured in a solution containing 20 mm MOPS-KOH (pH 7.0), 100 mm KCl, 1 mm MgCl2, 1 mm DTT, 0.25 mg/ml BSA, 12 μm CaM, 0.5 mm ATP, 2.5 mm phosphoenol pyruvate, 20 units/ml pyruvate kinase, 40 μm actin, 1 mm EGTA, and 0.02–0.04 μm Myo5a-FL. EGTA (1 mm) was replaced with 0.9 mm EGTA and 1 mm CaCl2 for pCa4 conditions. The values are means ± S.D. from three independent assays of three protein preparations. B, stimulation of the ATPase activity of Myo5a-FL by Mlph-GTBDP. The ATPase activity was measured in a solution containing 20 mm MOPS-KOH (pH 7.0), 200 mm NaCl, 1 mm MgCl2, 1 mm DTT, 0.25 mg/ml BSA, 12 μm CaM, 0.5 mm ATP, 2.5 mm phosphoenol pyruvate, 20 units/ml pyruvate kinase, 40 μm actin, 1 mm EGTA, 0.03–0.04 μm Myo5a-FL, and various concentrations of the Mlph-GTBDP. Stimulation of the ATPase activity of Myo5a-FL by Mlph-GTBDP was fit to a hyperbola, defining Kd, the apparent dissociation constants of Mlph-GTBDP to Myo5a-FL, which were 32.6 ± 8.2 μm for wild type and 7.3 ± 0.9 μm for R1490A/K1491A. The values are means ± S.D. from three independent assays of three protein preparations. C, GST pulldown of GST-Mlph-GTBDP with FLAG-tagged Myo5a-FL. The pulled down samples and the inputs were analyzed by SDS-PAGE and detected by Western blot using anti-FLAG antibody and anti-GST antibody (top panel). The amounts of pulled down Myo5a-FL were quantified using NIH image program. The bottom panel shows the means ± S.D. of the amount of pulled down Myo5a-FL (relative to WT) from three independent assays of a single protein preparation. D, GST pulldown of GST-Mlph-GTBDP with His-Myo5a-T1235-WT and His-Myo5a-T1344-WT or -RKAA. The pulled down samples were analyzed by SDS-PAGE and detected by Coomassie Blue staining (top panel). The amounts of pulled down His-Myo5a-T1235 and His-Myo5a-T1344 were quantified as described under “Experimental Procedures.” The bottom panel shows the means ± S.D. of the mole of pulled down His-Myo5a-T1344 (relative to His-Myo5a-T1235-WT) from five independent assays of a two protein preparations. RKAA, R1490A/K1491A; IB, immunoblotting.