Chemical Inhibitors of Myosin Function

Blebbistatin是在筛选非肌球蛋白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细胞分裂。我们测试了它对许多其他常规和非常规肌球蛋白异构体的活性。Blebbistatin抑制肌动蛋白激活的NMIIA重肌球蛋白（HMM）、NMIIB （HMM）、平滑肌HMM和几种横纹肌HMM的MgATPase活性。对肌动蛋白激活的MgATPase的一半最大抑制所需的blebbistatin的量范围从兔骨骼肌HMM的0.4微摩尔到平滑肌HMM的80微摩尔。有趣的是，即使这些分子与平滑肌肌球蛋白密切相关，3-5微摩尔blebbistatin也能最大限度地抑制NMIIA和NMIIB的一半。即使在100微摩尔浓度下，Blebbistatin也不能有效抑制大鼠myo1b、棘阿米巴肌球蛋白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-phalloidin标记的F-actin的运动。在洗掉blebbistatin后，这种效果是可逆的。蓝光（488 nm）照射可破坏blebbistatin的抑制活性。这一性质可用于研究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。抑制作用是复杂的，可能涉及到blebbistatin与肌球蛋白核苷酸复合物的结合，该复合物随后在原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.