BIOCHEMISTRY OF CONTRACTILE PROTEINS

我们的长远目标是了解监管机制(S) 在光滑的肌肉中。平滑肌对许多过程都是至关重要的 (例如，动、静脉)，用于治疗功能异常 对基础生物化学的欣赏是必不可少的。加价 在细胞内钙离子诱导收缩，这涉及到 肌球蛋白20,000道尔顿轻链的磷酸化 钙调蛋白依赖的肌球蛋白轻链激酶(MLCK)。 去磷酸化发生在松弛过程中。我们的数据表明 磷酸化通过改变细胞内的 肌球蛋白的构象(从非活性状态到活性状态)和 在生理条件下发生的变化 也存在于单体肌球蛋白的6S-10S转变过程中。这其中的一部分 因此，体外移植可以作为体内移植的模型。 进程。我们最初的目标是挑战形体活动 假设并证实酶活性是由 构象。接下来的重点将是确定那些 肌球蛋白分子中修饰ATPase的变化 活性和肌动蛋白结合，以定义在 10S-6S转换并将其与变化相关联 由磷酸化诱导。由于人们对此知之甚少 肌球蛋白分子结构的初步研究 三元和四元结构的表征是必不可少的。 这些可能履行监管职能的变化将是 已确认身份。影响肌球蛋白的一些因素 还将研究构象，包括肌动蛋白和 原肌球蛋白，目的是联系改变的构象 对生物特性的改良。它也将是 确定肌球蛋白构象是否能直接或影响动力学 与磷酸化和ATPase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 这些磷酸化的后果还没有确定，而且 这将被研究并与构象相关。我们的研究 关于MLCK的讨论将继续，我们将探讨 用有限的蛋白水解法组织分子并测试 脱辅酶因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和 将测试与钙离子无关的抑制活性 MLCK的形式。Caldesmon被认为是一种调节蛋白质 将进行研究，以评估这一点 假设。

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.