CALCIUM & MYOSIN LIGHT CHAIN KINASE IN HUMAN AORTA

肌球蛋白轻链激酶（MLCK）催化20个肌球蛋白的磷酸化。 kDa肌球蛋白调节轻链。在血管平滑肌中，MLCK起着 在最后的共同途径中起着关键的调节作用， 细胞内Ca 2+瞬变引起收缩起始。先前 对鸡平滑肌和兔骨骼肌MLCK的研究表明， 在钙调素结合区域内有一个区域与 20 kDa轻链的磷酸化区域。在不存在 Ca 2 +-钙调蛋白复合物，MLCK是无活性的，因为一部分的钙调蛋白复合物， 钙调素结合区特异性地与蛋白质底物相互作用 结合位点以这种抑制剂为模型的合成肽 MLCK的“假底物”区域有效地阻止了 肌球蛋白轻链竞争的方式。模拟的药理学药物 对假底物-MLCK活性位点的相互作用可能是特异性的 MLCK的抑制剂。本提案是工作的初始阶段 其目标是开发这种药物。Western印迹法 证实了从人主动脉分离人MLCK cDNA的可行性 使用抗鸡MLCK抗体构建文库。该图书馆的建设 lambda gt 11正在进行中。一个编码人血管平滑肌的cDNA 将对MLCK进行克隆和测序。一种活性钙调素调节片段 将其亚克隆到细菌表达载体中，表达， 提纯合成肽、缺失诱变和位点特异性 诱变将用于精确地定义钙调蛋白结合， 假底物域如果这种细菌的晶体结构 表达的片段被确定，该信息将用于设计 进一步的诱变实验。这些研究将导致一个更完整的 了解MLCK和Ca 2 +-钙调蛋白复合物的相互作用， 并可能导致一类新的药物的发展，用于治疗 以血管阻力增加为特征的疾病状态。

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.