CALCIUM AND CALMODULIN IN SMOOTH MUSCLE CONTRACTION

钙调蛋白(CaM)是一种普遍存在的钙结合蛋白，具有 作为钙信号的通用调节器出现。AS 细胞内钙升高，钙与钙调素结合产生结构变化 这使得它可以绑定和激活超过30种不同的 靶蛋白。钙调素直接参与钙依赖 调节平滑肌收缩、腺体和 神经分泌、DNA复制、基因表达和细胞 组织。目前的研究将在生物学上利用我们的新技术 有选择性地报告与CaM结合的活性荧光摄像头 它的目标蛋白。我们将确定并关联案例 Smooth中CaM与靶蛋白结合的依赖性 肌肉对钙的依赖使其被激活。vbl.使用 荧光停流技术，我们将确定速率 钙调素与其钙依赖性结合和解离的研究 靶蛋白，包括肌球蛋白轻链激酶(MLCK)和 卡尔德蒙。 这些比率将与钙的减少量进行比较 CaM-靶蛋白复合体。这些研究将提供 与钙瞬变相关的时间信息(例如 肌肉)与CaM-靶蛋白的结合率和 分离。 大多数外部刺激通过以下方式影响细胞内事件 细胞内激酶的激活与特异性蛋白的磷酸化 底物蛋白质。最近，有几项研究表明， 酪氨酸激酶(包括胰岛素受体和癌基因 产物，P60-src)能够磷酸化酪氨酸残基 在CaM的钙结合位点III和IV。我们将研究其影响 这种磷酸化对CaM的钙(和药物)结合的影响 CaM与And相互作用的性质及其钙依赖性 激活其靶蛋白。如果是磷酸化的CaM 显示改变了MLCK的钙依赖激活，它将是 检测其调节钙依赖张力的能力 化学剥离的冠状动脉。因为卡姆规定了 细胞新陈代谢和生长的许多方面及其磷酸化 通过这些酪氨酸激酶可能在 胰岛素作用机制及其在细胞转化中的作用 由编码酪氨酸激酶的致癌病毒所致。

Calmodulin (CaM) is a ubiquitous Ca binding protein that has emerged as a universal regulator of the calcium signal. As cytosolic Ca rises, Ca binds to CaM producing structural changes which allow it to bind and activate more than thirty different target proteins. CaM is directly involved in the Ca dependent regulation of smooth muscle contraction, glandular and neurosecretion, DNA replication, gene expression and cell division. The present studies will utilize our new biologically active fluorescent CaM's which selectively report CaM binding to its target proteins. We will determine and relate the Ca dependence of CaM binding to its target proteins in smooth muscle to the Ca dependence of their activation. Using fluorescence stopped-flow techniques we will determine the rates of the Ca dependent association and dissociation of CaM with its target proteins, including myosin light chain kinase (MLCK) and caldesmon. These rates will be compared to the off-rates of calcium from CaM-target protein complexes. These studies will provide temporal information relating Ca transients (such as those muscle) with the rate of CaM-target protein association and disassociation. Most external stimuli influence intracellular events by the activation of cellular kinases and the phosphorylation of specific substrate proteins. Recently, it has been shown that several tyrosine kinases (including, the insulin receptor and the oncogene product, P60-src) are capable of phosphorylating tyrosine residues in Ca binding sites III and IV of CaM. We will examine the effect of this phosphorylation on CaM's calcium (and drug) binding properties and on the Ca dependence of CaM interaction with and activation of its target proteins. If phosphorylated CaM exhibition altered Ca dependent activation of MLCK, it will be examined for its ability to regulate Ca dependent tension in chemically skinned coronary arteries. Because CaM regulates many aspects of cell metabolism and growth its phosphorylation by these tyrosine kinases might play a pivotal role in the mechanism of action of Insulin and in the transformation of cells by oncogenic viruses which code for tyrosine kinases.