BIOCHEMISTRY OF CONTRACTILE PROTEINS

我们的长期目标是了解监管机制 在平滑肌中。 平滑肌对许多过程至关重要 （例如：在动脉和静脉中）并且为了治疗异常功能 对基本生物化学的理解是必不可少的。 增加 在细胞内Ca 2+诱导收缩，这涉及到 肌球蛋白的20，000-道尔顿轻链的磷酸化 钙调蛋白依赖性肌球蛋白轻链激酶（MLCK）。 去磷酸化发生在松弛状态。 我们的数据显示， 磷酸化通过改变细胞膜上的 肌球蛋白的构象（从非活性状态到活性状态）， 在生理条件下发生的变化 单体肌球蛋白的6S-10 S转变也是如此。 份该 因此，体外转化可以作为体内转化的模型。 过程 我们最初的目标是挑战 假设，并证实酶活性是由 构象 随后的重点将是确定那些 改变ATP酶的肌球蛋白分子中发生的变化 活动和肌动蛋白结合，以定义发生的变化， 10 S-6S转换，并将这些与改变相关联， 由磷酸化诱导。 由于对这一问题知之甚少， 平滑肌肌球蛋白分子结构的初步研究 三级和四级结构的表征是必要的。 这些可能履行监管职能的变化将是 鉴定 一些影响肌球蛋白的因素 构象也将进行研究，包括肌动蛋白和 原肌球蛋白，目的是将改变的构象 to a modificationof biological生物properties属性. 还将 确定肌球蛋白构象是否可以指导或影响动力学 与磷酸化和ATP酶相关的参数 活动 轻链上的其他位点被磷酸化 通过MLCK和蛋白激酶C。 然而，生物 这些磷酸化的后果尚未确定， 这将被研究并与构象相关联。 我们的研究 将继续进行MLCK，我们将探索功能 组织的分子使用有限的蛋白水解和测试 脱辅基酶由于相互作用而失活的假说 有一个抑制区 合成肽，基于已知的 钙调蛋白结合位点的序列，将被用作模型， 将测试与Ca 2+非依赖性 MLCK的形式。 Caldesmon被认为是一种调节蛋白， 平滑肌和研究将进行评估， 假说.

Our long range goal is to understand the regulatory mechanism(s) in smooth muscle. Smooth muscle are vital for many processes (eg. in arteries and veins) and in order to treat abnormal function an appreciation of the basic biochemistry is essential. An increase in intracellular Ca2+ induces contraction and this involves phosphorylation of the 20,000-dalton light chain of myosin via the calmodulin-dependent myosin light chain kinase (MLCK). Dephosphorylation occurs in relaxation. Our data suggests that phosphorylation controls contractile activity by changing the conformation of myosin (from an inactive to an active state) and that the changes occurring under physiological conditions occur also in the 6S-10S transition of monomeric myosin. Parts of this in-vitro transition therefore can serve as a model for the in-vivo process. Our initial objective is to challenge the shape-activity hypothesis and to confirm that enzymatic activity is directed by conformation. A subsequent emphasis will be to identify those changes that occur in the myosin molecule that modify ATPase activity and actin-binding, to define the changes that occur during the 10S-6S transition and to correlate these to the alterations induced by phosphorylation. Since little is known about the structures of the smooth muscle myosin molecule a preliminary characterization of tertiary and quaternary structure is essential. Those changes that might fulfil a regulatory function will be identified. Some of the factors that influence myosin conformation will also be studied, including actin and tropomyosin, with the objective of relating altered conformation to a modificaiton of biological properties. It will also be determined if myosin conformation can direct or influence kinetic parameters associated with phosphorylation and with ATPase activities. Additional sites on the light chain are phosphorylated by both MLCK and protein kinase C. However, the biological consequences of these phosphorylations are not established and this will be studied and correlated to conformation. Our studies on MLCK will be continued and we will probe the functional organization of the molecule using limited proteolysis and test the hypothesis that the apoenzyme is inactive because of interaction with an inhibitory zone. Synthetic peptides, based on the known sequences of calmodulin-binding sites, will be used as models and will be tested for inhibitory activity with the Ca2+-independent form of MLCK. Caldesmon is proposed as a regulatory protein in smooth muscle and studies will be carried out to evaluate this hypothesis.