Pharmacological Response of Smooth Muscle is related With its Myosin Phosphorylation.

NA0344是一种新型的抗生素衍生物，它能抑制含有磷酸酶和钙调蛋白/肌球蛋白轻链(MLCK)活性的20,000 mR肌球蛋白轻链(LC)的磷酸化(IC_(50)=6.2×10~(-6)g/ml)。NA0344还抑制肌球蛋白与肌球蛋白的相互作用，超沉淀法和ATPase活性检测结果分别为3.7x10；-7&gt；和3.8×10；-7&gt；g/ml。NA0344部分抑制肌球蛋白磷酸化的肌球蛋白包括未磷酸化的肌球蛋白、具有未磷酸化LC和磷酸化LC的异二聚体肌球蛋白以及具有LC磷酸化的同源二聚体肌球蛋白。我们建立了分离这些异构体的分析电泳法，发现天然肌球蛋白中的同源二聚体的IC_(50)为10~(-7)g/ml。如果我们假设只有同源二聚体在肌动蛋白-肌球蛋白-ATP相互作用中是活跃的，那么这种相互作用的抑制可以通过肌球蛋白与MLCK的磷酸化来解释。最近，冈田酸被报道增强了肌动蛋白-肌球蛋白-ATP的相互作用。这种作用被解释为通过抑制磷酸酶活性而促进磷酸化而起作用。我们成功地制备了不含磷酸酶活性的天然肌球蛋白。使用该制剂，观察到了增强作用，表明冈田酸直接通过肌球蛋白增强了相互作用。

NA0344, a novel derivative of antibiotics, inhibited phosphorylation of 20,000 Mr myosin light chain (LC) of smooth muscle native actomyosin containing both phosphotase and calmodulin/myosin LC kinas (MLCK) activity (IC_<50> = 6.2 x 10^<-6> g/ml). The inhibitory effect is attributable to the direct inhibition of MLCK.NA0344 also inhibited actin-myosin-ATP interaction of the native actomyosin with IC_<50> = 3.7 x 10^<-7> and 3.8 x 10^<-7> g/ml as monitored by superprecipitation and ATPase activity, respectively.Thoretically, myosin whose phosphorylation is partially inhibited by NA0344 comprises from unphosphorylated myosin, heterodimer myosin isoform with an unphosphorylated LC and a phosphorylated LC, and homodimer myosin isoform with both LC phosphorylated. We have developed the analytical electrophoresis separating these isomers and found that IC_<50> for the homodimer in native actomyosin was at the level of 10^<-7> g/ml. If we assume that only the homodimer is active in actin-myosin-ATP interaction, inhibition of the interaction can be explatined by that of myosin phosphorylation with MLCK.Recently, okadaic acid has been reported to enhance actin-myosin-ATP interaction. The effect is interpreted to be mediated by inhibiting phosphatase activity resulting in the enhancement of phosphorylation. We succeeded in preparing the native actomyosin which does not contain phosphatase activity. With this preparation, the enhancing effect was observed, indicating that okadaic acid enhances the interaction directly through myosins.