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。Blebbistatin可抑制NMIIA重肌球蛋白（HMM）、NMIIB（HMM）、平滑肌HMM和几种横纹肌HLAs的肌动蛋白激活的MgATPase活性。对于肌动蛋白激活的MgATP酶的半数最大抑制所需的blebbistatin的量范围为从0.4微摩尔（对于兔骨骼肌HMM）到80微摩尔（对于平滑肌HMM）。有趣的是，NMIIA和NMIIB均被3-5微摩尔的blebbistatin抑制一半，即使这些分子与平滑肌肌球蛋白密切相关。即使在100微摩尔浓度下，Blebbistatin也不能有效抑制大鼠myo 1b、阿米巴肌球蛋白IC、肌球蛋白-V和肌球蛋白-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。用蓝光（488 nm）照射可破坏blebbistatin的抑制活性。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。我们对blebbistatin抑制肌球蛋白的动力学机制进行了初步的表征。这种抑制作用是复杂的，可能涉及blebbistatin与肌球蛋白核苷酸复合物的结合，该复合物随后在最初结合的核苷酸解离后抑制新ATP的结合。

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. Blebbistatin inhibited the actin-activated MgATPase activity of NMIIA heavy meromyosin (HMM), NMIIB (HMM), smooth muscle HMM, and several striated muscsle muscle HMMs. The amount of blebbistatin required for half maximal inhibition of the actin-activated MgATPase is ranged form 0.4 micromolar for rabbit skeletal muscle HMM to 80 micromolar for smooth muscle HMM. Interestingly NMIIA and NMIIB were both half maximally inhibited by 3-5 micromolar blebbistatin even though these molecules are closely related to smooth muscle myosin. Blebbistatin, even at 100 micromolar, did not effectively inhibit the activity of rat myo1b, Acanthamoeba myosin IC, 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. The inhibitory activity of blebbistatin was destroyed by illumination with blue light (488 nm). This property could be useful in studying the reversibility of blebbistatin inhibition of contractile processes in cells and for synchronizing contractile events. We have done a preliminary characterization of the kinetic mechanism for the blebbistatin inhibition of myosin. The inhibition is complex and probably involves blebbistatin binding to a myosin nucleotide complex which subsequently inhibits the binding of a new ATP after the dissociation of the originally bound nucleotide.