CALCIUM & MYOSIN LIGHT CHAIN KINASE IN HUMAN AORTA

肌球蛋白轻链激酶(MLCK)催化20 KDA调控肌球蛋白轻链。在血管平滑肌中，MLCK发挥作用 在最终的共同途径中发挥关键的调节作用 细胞内的钙瞬变产生收缩的启动。上一首 对鸡平滑肌和兔骨骼肌MLCK的研究表明 在钙调蛋白结合区内有一个与 20 kDa轻链的磷酸化区域。在缺席的情况下 钙调素复合体，MLCK是不活跃的，因为一部分 钙调蛋白结合区与蛋白质底物特异性相互作用 结合部位。以这种抑制作用为模型的合成肽 MLCK的“假底物”区可有效地阻止其磷酸化 肌球蛋白轻链以竞争的方式。模拟的药理制剂 关于假底物-MLCK活性部位的相互作用可能是特异的 MLCK的抑制剂。这项提案代表了工作的初始阶段 以开发这类药物为目标。西方的印迹已经 证实从人主动脉中分离人MLCK基因的可行性 文库采用抗鸡MLCK抗体。这个图书馆的建造是在 Lambda gt11正在进行中。编码人血管平滑肌的基因片段 MLCK将被克隆和测序。钙调素调节的活性片段 将被亚克隆到细菌表达载体中，表达，并 纯净的。合成肽、缺失突变和位点特异性 突变将被用来精确地定义钙调蛋白结合和 伪标记域。如果这种细菌的晶体结构 表达片段被确定，该信息将被用于设计 进一步的诱变实验。这些研究将导致更完整的 了解MLCK与钙-钙调蛋白复合体的相互作用， 并可能导致开发一类治疗糖尿病的新药 血管阻力增加的疾病状态。

Myosin light chain kinase (MLCK) catalyzes the phosphorylation of the 20 kDa regulatory light chain of myosin. In vascular smooth muscle, MLCK plays a critical regulatory role in the final common pathway through which intracellular Ca2+ transients produce initiation of contraction. Previous work with chicken smooth muscle and rabbit skeletal muscle MLCK have shown that within the calmodulin binding region is a region with similarity to the phosphorylated region of the 20 kDa light chain. In the absence of the Ca2+-calmodulin complex, MLCK is inactive because a portion of the calmodulin binding region specifically interacts with the protein substrate binding site. Synthetic peptides modeled on this inhibitory "pseudosubstrate" region of MLCK effectively prevent the phosphorylation of myosin light chain in a competitive fashion. Pharmacologic agents modeled on the pseudosubstrate-MLCK active site interaction may be specific inhibitors of MLCK. This proposal represents the initial phases of work directed toward the goal of developing such agents. Western blots have confirmed the feasibility of isolating a human MLCK cDNA from a human aorta library using anti-chicken MLCK antibodies. Construction of this library in lambda gt11 is in progress. A cDNA encoding human vascular smooth muscle MLCK will be cloned and sequenced. An active, calmodulin regulated fragment will be subcloned into a bacteria expression vector, expressed, and purified. Synthetic peptides, deletion mutagenesis and site specific mutagenesis will be used to precisely define the calmodulin binding and pseudosubstrate domains. If the crystal structure of this bacterially expressed fragment is determined, that information will be used to design further mutagenesis experiments. These studies will lead to a more complete understanding of the interaction of MLCK and the Ca2+-calmodulin complex, and may lead to the development of a new class of drugs for treatment of disease states characterized by increased vascular resistance.