Chemical Inhibitors Of Myosin Function

在筛选非肌肉肌球蛋白IIA(NMIIA)ATPase活性的化学抑制剂中发现了blbbistatin。它能抑制非洲爪哇和人类细胞的起泡和胞质分裂。我们测试了它与其他一些常规和非常规肌球蛋白亚型的活性。低浓度(100微摩尔)的Blebbistatin抑制NMIIA HMM、NMIIB Heavy Mermyosin(HMM)、平滑肌HMM和骨骼肌HMM的肌动蛋白激活的镁ATPase活性。肌动蛋白激活的镁ATPase的半数最大抑制所需的博莱菌素的量为0.5微摩尔，而对于平滑肌HMM则为50微摩尔。0.5M的Blebbistatin对骨骼HMM的肌动蛋白激活的镁ATPase抑制50%。灯盏花素，即使在100微摩尔，也不能有效地抑制myo1b、myosin-V和myosin-X的活性。灯盏花素完全抑制NMIIA和骨骼肌HMM标记的罗丹明-鬼臼毒素标记的F-肌动蛋白的运动。这种作用在被Blebbistatin洗掉后是可逆的。在100微摩尔的Blebbistatin存在下，用罗丹明激发(532 Nm)进行长时间(2-3分钟)的观察，肌动蛋白细丝在观察场中开始缓慢移动。随后将玻片移动到新的观察场将再次显示完全抑制运动性，这表明这种效果是由于Blebbistatin的光化学失活，而不是简单的依赖于时间的抑制丧失。Alexa-488-激动剂(488 Nm)可迅速灭活滤过泡抑素，使Alexa-488-phalloidin标记的肌动蛋白在显微镜下看不到运动的抑制。后一项实验是在488 nm光强度极低的情况下进行的，这表明blbbistatin对该波长非常敏感。这一特性可能有助于研究Blebbistatin抑制细胞收缩过程的可逆性，并用于同步收缩事件。

Blebbistatin was discovered in a screen for chemical inhibitors of nonmuscle myosin IIA (NMIIA) ATPase activity. It inhibits blebbing and cytokinesis in Xenopus and human cells. We tested its activity against a number of other conventional and unconventional myosin isoforms. At low ATP concentrations (<100 micromolar) blebbistatin inhibited the actin-activated MgATPase activity of NMIIA HMM, NMIIB heavy meromyosin (HMM), smooth muscle HMM, and skeletal muscle HMM. The amount of blebbistatin required for half maximal inhibition of the actin-activated MgATPase is 0.5 and 50 micromolar for smooth muscle HMM. The actin-activated MgATPase of skeletal HMM was inhibited by 50% at 0.5 ?M blebbistatin. Blebbistatin, even at 100 micromolar, did not effectively inhibit the activity of myo1b, myosin-V and myosin-X. Blebbistatin completely inhibited the movement of rhodamine-phalloidin labeled F-actin by NMIIA and skeletal muscle HMM. This effect was reversible upon wash out of blebbistatin. At long times of observation (2-3 minutes) using rhodamine-excitation (532nm) in the presence of 100 micromolar blebbistatin, actin filaments would begin to move slowly in the field of observation. Subsequent movement of the slide to a new observation field would again show complete inhibition of motility suggesting that the effect was due to photochemical inactivation of blebbistatin and not a simple time dependent loss of inhibition. Alexa-488-excitation (488 nm) rapidly inactivated the blebbistatin such that no inhibition of movement of Alexa-488-phalloidin-labeled actin was apparent in the microscope. The latter experiment was conducted under evanescent illumination where the intensity of 488 nm light was extremely low, suggesting that blebbistatin is markedly sensitive to this wavelength. This property could be useful in studying the reversibility of blebbistatin inhibition of contractile processes in cells and for synchronizing contractile events.