Fujita K, Kohama K
Department of Pharmacology, Gunma University School of Medicine, Japan.
Nihon Yakurigaku Zasshi. 1994 Nov;104(5):363-8. doi: 10.1254/fpj.104.363.
The sites of action of many chemical agents that modify the contraction of smooth muscle are in the smooth muscle membrane. However, a few agents, such as calmodulin inhibitors and protein kinase inhibitors, interact directly with contractile elements of the actomyosin system so as to modify smooth muscle contraction. Here, we describe experimental procedures that are applicable for the screening of smooth muscle relaxants with this mode of action. Myosin B was extracted from chicken gizzard smooth muscle. Because myosin B was a crude preparation of smooth muscle actomyosin, it consisted of regulatory proteins of calmodulin, myosin light chain kinase and protein phosphatase in addition to the contractile proteins of actin and myosin. Interaction of chemical agents with these proteins could be detected by measuring the Mg-ATPase activity of the myosin B preparation. Then we examined whether the agents that altered the ATPase activity was associated with changes in phosphorylation of myosin light chain. If the levels are altered, the agents may interact with the regulatory protein(s). If not, the site of their action was in the contractile proteins. The analysis with these respective proteins will be also described.
许多能改变平滑肌收缩的化学制剂的作用位点在平滑肌膜上。然而，一些制剂，如钙调蛋白抑制剂和蛋白激酶抑制剂，直接与肌动球蛋白系统的收缩成分相互作用，从而改变平滑肌收缩。在此，我们描述了适用于筛选具有这种作用方式的平滑肌松弛剂的实验程序。肌球蛋白B从鸡砂囊平滑肌中提取。由于肌球蛋白B是平滑肌肌动球蛋白的粗制品，除了肌动蛋白和肌球蛋白的收缩蛋白外，它还包含钙调蛋白、肌球蛋白轻链激酶和蛋白磷酸酶等调节蛋白。化学制剂与这些蛋白质的相互作用可以通过测量肌球蛋白B制剂的镁 - ATP酶活性来检测。然后我们检查改变ATP酶活性的制剂是否与肌球蛋白轻链的磷酸化变化有关。如果水平发生改变，这些制剂可能与调节蛋白相互作用。如果没有改变，它们的作用位点就在收缩蛋白中。还将描述对这些各自蛋白质的分析。
