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

NA0344是一种新型抗生素衍生物，可抑制含有磷酸酶和钙调蛋白/肌球蛋白LC激酶（MLCK）的平滑肌天然肌动球蛋白的20,000 Mr肌球蛋白轻链（LC）的磷酸化活性（IC_<50> = 6.2 × 10^<-6> g/ml）。这种抑制作用可归因于MLCK的直接抑制作用。NA0344对肌动蛋白-肌球蛋白- atp的相互作用也有抑制作用，IC_<50> = 3.7 × 10^<-7>和3.8 × 10^<-7> g/ml。理论上，被NA0344部分抑制磷酸化的肌球蛋白包括未磷酸化的肌球蛋白、具有未磷酸化的LC和磷酸化的LC的异二聚体肌球蛋白异构体，以及具有两个LC磷酸化的同二聚体肌球蛋白异构体。我们开发了分离这些异构体的分析电泳，发现天然肌动球蛋白中同型二聚体的IC_<50>在10^<-7> g/ml的水平。如果我们假设只有同型二聚体在肌动蛋白-肌球蛋白- atp相互作用中有活性，那么这种相互作用的抑制可以用肌球蛋白与MLCK的磷酸化来解释。最近，冈田酸被报道增强肌动蛋白-肌球蛋白- atp的相互作用。这种效应被解释为通过抑制磷酸酶活性导致磷酸化增强而介导的。我们成功地制备了不含磷酸酶活性的天然肌动球蛋白。用该制剂观察到增强作用，表明冈田酸直接通过肌球蛋白增强相互作用。

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.