Chemical Inhibitors Of Myosin Function

Blebbistatin是在筛选非肌肉肌球蛋白IIA（NMIIA）ATP酶活性的化学抑制剂时发现的。它抑制非洲爪蟾和人类细胞的起泡和胞质分裂。我们测试了它对其他一些常规和非常规肌球蛋白亚型的活性。在低ATP浓度（<100微摩尔）下，blebbistatin抑制NMIIA HMM、NMIIB重酶解肌球蛋白（HMM）、平滑肌HMM和骨骼肌HMM的肌动蛋白活化MgATP酶活性。对于平滑肌HMM，肌动蛋白激活的MgATP酶的半数最大抑制所需的blebbistatin的量为0.5和50微摩尔。肌动蛋白激活的MgATPase的骨骼HMM抑制50%，在0.5？M blebbistatin。即使在100微摩尔浓度下，Blebbistatin也不能有效抑制myo 1b、myosin-V和myosin-X的活性。Blebbistatin完全抑制NMIIA和骨骼肌HMM对罗丹明-鬼笔环肽标记的F-actin的运动。这种作用在blebbistatin洗脱后是可逆的。在100微摩尔的blebbistatin存在下，使用罗丹明激发（532 nm）进行长时间的观察（2-3分钟），肌动蛋白丝将开始在观察视野中缓慢移动。随后将载玻片移动到新的观察视野将再次显示运动性的完全抑制，表明该效应是由于blebbistatin的光化学失活，而不是简单的时间依赖性抑制丧失。Alexa-488-激发（488 nm）快速灭活blebbistatin，使得Alexa-488-鬼笔环肽标记的肌动蛋白的运动在显微镜下没有明显的抑制。后一个实验在488 nm光强度极低的渐逝照明下进行，表明blebbistatin对该波长显著敏感。这一特性可能有助于研究blebbistatin抑制细胞收缩过程的可逆性和同步收缩事件。

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.