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

NA 0344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）活性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（IC_<50>= 6.2 × 10 - 6 μ <-6>g/ml）。NA 0344还抑制天然肌动球蛋白的肌动蛋白-肌球蛋白-ATP相互作用，通过超沉淀和ATP酶活性监测，IC_（max）分别<50>为3.7 × 10 ~（-6）μ g/ml<-7>和3.8 × 10 ~（-6）μ g/ml<-7>。和均为LC磷酸化的同型二聚体肌球蛋白同种型。我们建立了分离这些异构体的分析电泳法，发现<50>天然肌动球蛋白中同二聚体的IC_（10 μ <-7>g/ml）水平。如果我们假设只有同二聚体在肌动蛋白-肌球蛋白-ATP相互作用中是有活性的，那么这种相互作用的抑制可以解释为肌球蛋白被MLCK磷酸化。该效应被解释为通过抑制磷酸酶活性介导，导致磷酸化增强。我们成功地制备了不含磷酸酶活性的天然肌动球蛋白。使用该制剂，观察到增强效应，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.