Trybus K M, Krementsova E, Freyzon Y
Department of Physiology and Molecular Biophysics, University of Vermont, Burlington, Vermont 05405, USA.
J Biol Chem. 1999 Sep 24;274(39):27448-56. doi: 10.1074/jbc.274.39.27448.
An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulin-binding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 +/- 1.6 s(-1) with a K(app) of approximately 1 microM (37 degrees C), and the velocity of actin movement was approximately 0.3 micrometer/s (30 degrees C). Calcium inhibited both of these activities, but the addition of calmodulin restored the values to approximately 70% of control, indicating that calmodulin dissociation caused inhibition. In contrast to myosin II, MD(2IQ) is highly associated with actin at physiological ionic strength in the presence of ATP, but the motor is in a weakly bound conformation based on the pyrene-actin signal. The rate of dissociation of acto-MD(2IQ) by ATP is fast (>850 s(-1)), and ATP hydrolysis occurs at approximately 200 s(-1). The affinity of acto-MD(2IQ) for ADP is somewhat higher than that of smooth S1, and ADP dissociates more slowly. Actin does not cause a large increase in the rate of ADP release, nor does the presence of ADP appreciably alter the affinity of MD(2IQ) for actin. These kinetic data suggest that monomeric myosin V is not processive.
一种由运动结构域和两个钙调蛋白结合IQ模体组成的表达型单体鼠肌球蛋白V构建体（MD(2IQ)）被用于评估这种非常规肌球蛋白的调节和动力学特性。在乙二醇双醚四乙酸（EGTA）存在的情况下，MD(2IQ)的肌动蛋白激活的ATP酶活性为7.4±1.6 s⁻¹，其表观解离常数（K(app)）约为1微摩尔（37摄氏度），肌动蛋白运动速度约为0.3微米/秒（30摄氏度）。钙抑制了这两种活性，但添加钙调蛋白后将这些值恢复到对照值的约70%，表明钙调蛋白解离导致了抑制作用。与肌球蛋白II不同，在ATP存在的生理离子强度下，MD(2IQ)与肌动蛋白高度结合，但基于芘-肌动蛋白信号，该运动蛋白处于弱结合构象。ATP使肌动蛋白-MD(2IQ)解离的速率很快（>850 s⁻¹），ATP水解发生的速率约为200 s⁻¹。肌动蛋白-MD(2IQ)对ADP的亲和力略高于平滑肌肌球蛋白S1，且ADP解离更慢。肌动蛋白不会导致ADP释放速率大幅增加，ADP的存在也不会明显改变MD(2IQ)对肌动蛋白的亲和力。这些动力学数据表明单体肌球蛋白V不是持续性的。
