ROLE OF PHOSPHORYLATION AS A REGULATORY MECHANISM IN MUSCLE CONTRACTION

肌球蛋白和非肌球蛋白的磷酸化调控 20 kDa调控轻链(RLC)对 血管平滑肌收缩与肌球蛋白功能的关系 非肌肉细胞。磷酸化的部位在颈区 肌球蛋白，与ATP和肌动蛋白结合部位相距甚远 肌球蛋白。磷酸化位点位于现在所指的区域内 作为肌球蛋白的调节域，它由一个α-螺旋组成 重链的伸展，形成必需的结合部位 轻链(ELC)和RLC。重链本身就会发生二聚化 羧基末端到RLC结合部位，并开始形成棒状 一份。因此，在这个颈部区域，有六条多肽链 且两者之间相互作用是可能的 对于监管来说，脖子可能很重要。我们为此提供证据 通过证明蛋白水解法制备的单头肌球蛋白 移除两个头部中的一个不再受磷酸化的调节， 而是在本质上是活跃的。法规的另一个方面 平滑和非肌肉肌球蛋白的最大特点是它们能够采用折叠的 沉降系数为10s的结构。这里，尾巴 肌球蛋白区域循环回到自己身上，似乎与 监管领域区域。形成10S构象的能力 受磷酸化调节，并被认为参与了 调节细丝组装，可能是一种进一步 抑制未磷酸化的平滑肌酶活性 肌球蛋白。我们将肌球蛋白在10S构象中进行了交联化。一个 RLC的Cys-108与Cys-108形成共价交联 肌球蛋白重链尾巴的一部分，与调节分子接触 折叠过程中的域。交联型肌球蛋白在以下情况下不能展开 磷酸化或暴露在高离子强度条件下。我们的 结果表明，交联型折叠的肌球蛋白即使在 RLCs被磷酸化。

The regulation of smooth muscle and nonmuscle myosin by phosphorylation of the 20 kDa regulatory light chain (RLC) is of profound importance to contraction of smooth muscle tissue and the function of myosin in nonmuscle cells. The site of phosphorylation is in the neck region of myosin and is quite remote from the ATP and actin binding sites on myosin. The phosphorylation site lies within an area now referred to as the regulatory domain of myosin which consists of an alpha-helical stretch of heavy chain which forms the binding sites for the essential light chain (ELC) and RLC. The heavy chain dimerizes with itself just carboxyl-terminal to the RLC binding site and begins to form the rod portion. Thus, at this neck region, there are six polypeptide chains in close proximity and it is possible that interaction between the two necks might be important for regulation. We provide evidence for this by demonstrating that single-headed myosin prepared by proteolytically removing one of the two heads is no longer regulated by phosphorylation, but rather is constitutively active. Another aspect of the regulation of smooth and nonmuscle myosins is their ability to adopt a folded structure with a sedimentation coefficient of 10S. Here, the tail region of myosin loops back upon itself and appears to make contact with the regulatory domain region. The ability to form the 10S conformation is regulated by phosphorylation and is thought to be involved in the regulation of filament assembly and maybe a mechanism for further suppressing the enzymatic activity of unphosphorylated smooth muscle myosin. We cross-linked myosin while in the 10S conformation. A covalent cross-link was formed between Cys-108 of the RLC and the portion of the myosin heavy chain tail that contacts the regulatory domain during folding. The cross-linked myosin could not unfold when phosphorylated or when exposed to high ionic strength conditions. Our results show that the cross-linked, folded myosin is inactive even when the RLCs are phosphorylated.