ACTIN BASED REGULATION OF SMOOTH MUSCLE CONTRACTION

对Smooth中力产生机制的认识 肌肉及其调节在处理非常重要的 与血管等平滑肌相关的严重疾病 疾病。当完全理解时，流畅之间的差异 骨骼肌可以通过治疗来对抗 这些障碍。平滑的肌肉会产生更大的张力 肌球蛋白分子比骨骼肌更重要。平滑的肌肉可以 刺激后也要长时间保持紧张 (闩锁)。这些差异可能是由于 亚细胞组织，肌球蛋白的结构，以及 监管体系。监管制度是特殊的 医学上的重要性，因为只有它们才能被 外部影响。的一个特别有趣的组件 平滑肌的调节系统是肌动蛋白和钙调蛋白 结合蛋白钙调蛋白。 Caldesmon通过抑制肌动蛋白激活的ATP水解酶 光滑和骨骼肌球蛋白及其蛋白水解亚片段。 骨骼肌球蛋白ATPase活性的抑制 亚片段-1与抑制结合有关 亚片段-1到肌动蛋白。相比之下，重型猪的ATPase活性 来自平滑肌的肌球蛋白随着血管内皮细胞中 重肌球蛋白与肌动蛋白的结合。目前的建议是 确定caldesmon如何对绑定产生相反的影响 并且仍然产生对ATPase活性的抑制。在这样做的过程中 钙调蛋白的活性机制将被阐明，并进一步 本课程将探讨平滑肌球蛋白和骨骼肌球蛋白之间的区别。 光滑重物结合力增强的三个假设 将探索在有ATP存在的情况下的肌球蛋白：(1) 肌动蛋白-钙调蛋白-1上存在两个结合位点 另一种是非生产性的。(2)ADP率的降低 放手。(3)肌动蛋白的钙依赖性交联 细丝。这些研究将涉及对以下物质结合的测量 钙调蛋白和肌球蛋白亚段到肌动蛋白，稳态 动力学，使用停流进行快速动力学测量 分光光度计，化学交联型肌动蛋白、肌球蛋白和 钙调蛋白，各种蛋白质-蛋白质的蛋白分解消化 确定相互作用部位和亲和力的络合物 层析法。更多的研究旨在确定 未鉴定的钙调蛋白结合蛋白在细胞周期中的作用 监管流程。此外，反转的机制 钙调蛋白的抑制活性将被研究。

An understanding of the mechanism of force production in smooth muscle and its regulation is essential in dealing with the very serious disorders associated with smooth muscle such as vascular disease. When fully understood, the differences between smooth and skeletal muscles can be exploited therapeutically to combat these disorders. Smooth muscle generates more tension per myosin molecule than does skeletal muscle. Smooth muscle can also maintain tension for long periods of time after stimulation (latch). These differences are probably due to differences in the subcellular organization, the structure of myosin, and the regulatory systems. The regulatory systems are of particular medical importance since only they can be modulated by an external influence. A particularly interesting component of the regulatory system of smooth muscle is the actin and calmodulin binding protein caldesmon. Caldesmon inhibits the actin activated hydrolysis of ATP by smooth and skeletal myosin and their proteolytic subfragments. The inhibition of the ATPase activity of skeletal myosin subfragment-1 is associated with an inhibition of the binding of subfragment-1 to actin. In contrast, the ATPase activity of heavy meromyosin from smooth muscle occurs with an increase in the binding of heavy meroymosin to actin. The present proposal is to determine how caldesmon can have opposite effects on binding and still produce inhibition of ATPase activity. In so doing the mechanism of caldesmon activity will be clarified and further differences between smooth and skeletal myosins will be explored. Three hypotheses for the increased binding of smooth heavy meromyosin in the presence of ATP will be explored: (1) The presence of two binding sites on actin-caldesmon - one productive and the other nonproductive. (2) The reduction in the rate of ADP release. (3) A calcium dependent crosslinking of the actin filaments. These studies will involve measurement of binding of caldesmon and myosin subfragments to actin, steady-state kinetics, rapid kinetic measurements using a stopped-flow spectrophotometer, chemically crosslinking actin, myosin and caldesmon, proteolytic digestion of various protein-protein complexes to determine sites of interaction, and affinity chromatography. Additional studies are designed to determine the role of uncharacterized caldesmon binding proteins in the regulatory process. Also, the mechanism of reversal of caldesmon's inhibitory activity will be investigated.