Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin and Nonmuscle Myosin-2
Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin and Nonmuscle Myosin-2
复制标题
布雷他汀抑制平滑肌肌球蛋白和非肌肉肌球蛋白-2 的表征
DOI：
10.1021/acs.biochem.7b00311
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发表时间：
2017
期刊：
影响因子：
2.9
通讯作者：
Li Xiang-dong
中科院分区：
文献类型：
--
作者：
Zhang Hai-Man;Ji Huan-Hong;Ni Tong;Ma Rong-Na;Wang Aibing;Li Xiang-dong
Blebbistatin是II类肌球蛋白（包括横纹肌肌球蛋白和非肌肉肌球蛋白-2（NM 2））运动功能的有效和特异性抑制剂。然而，尚未评估NM 2c的blebbistatin抑制作用，并且关于其与平滑肌肌球蛋白（SmM）的疗效仍存在争议，SmM与NM 2高度同源。为了澄清这些问题，我们分析了blebbistatin对重组SmM和三种NM 2（NM 2a，-2b和-2c）的运动活性的影响。结果表明，Blebbistatin对SmM、NM 2s、NM 2b和NM 2c的ATP酶活性均有明显的抑制作用，其IC 50分别为6.47 μM、3.58 μM、2.30 μM和1.57 μM。为了鉴定抗blebbistatin的肌球蛋白-2突变体，我们对SmM和NM 2的blebistatin结合位点中的保守残基进行了诱变分析。我们发现A456 F突变使SmM和NM 2s对blebbistatin具有抗性，而不会大大改变它们的运动活性或磷酸化依赖性调节，使A456 F成为研究NM 2s细胞功能的有用突变体。
Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been assessed and remains controversial with respect to its efficacy with smooth muscle myosin (SmM), which is highly homologous to NM2. To clarify these issues, we analyzed the effects of blebbistatin on the motor activities of recombinant SmM and three NM2s (NM2a, -2b, and -2c). We found that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC50values: 6.47 μM for SmM, 3.58 μM for NM2a, 2.30 μM for NM2b, and 1.57 μM for NM2c. To identify the blebbistatin-resistant myosin-2 mutant, we performed mutagenesis analysis of the conserved residues in the blebbistatin-binding site of SmM and NM2s. We found that the A456F mutation renders SmM and NM2s resistant to blebbistatin without greatly altering their motor activities or phosphorylation-dependent regulation, making A456F a useful mutant for investigating the cellular function of NM2s.