STRUCTURE VS. FUNCTION IN MYOSIN LIGHT CHAIN KINASE

本项目的长期目标是更好地了解 平滑肌收缩的生理学和生物化学。调控 平滑肌收缩是由钙离子-钙调蛋白依赖性酶肌球蛋白引起的 轻链激酶（MLCK），磷酸化 肌球蛋白磷酸化是肌动蛋白激活肌球蛋白的先决条件 ATP酶和收缩。有人建议MLCK包含一个 由钙调素结合调节的抑制区。MLCK也 含有催化区和肌动蛋白结合区。的功能 羧基端（约24 kDa）未知，但初步 有证据表明，这一部分的表达独立于MLCK。的 分离该酶的部分cDNA使得可以使用 分子生物学技术，以进一步了解这一领域的定义 这些结构域的结构与功能之间的关系。这 cDNA是60%完整的，包括羧基末端，但 分子的氨基末端是未知的。具体目标， 提出了以下几点建议：1）建立一个表达活性的细菌系统 和Ca 2 +-钙调蛋白依赖性酶的部分cDNA; 2）定义 使用定点和缺失技术， 突变; 3）通过分离MLCK，确定MLCK的全长序列。 延伸部分cDNA的5 '末端的cDNA克隆;和4）表征 一种新的酸性蛋白（24 kDa），已从平滑肌中分离出来， 被认为与MLCK的羧基末端相同。 MLCK是平滑肌中的关键调节组分，并且是平滑肌细胞中的一种明确的调节因子。 了解其机制对我们理解正常的 平滑肌功能这是治疗异常的前提 平滑肌行为;一个重要的例子是血管平滑肌。 这些研究将有助于设计药物， 功能异常的治疗。

The long-term goal of this project is to gain a better understanding of the physiology and biochemistry of smooth muscle contraction. Regulation of smooth muscle contraction is by the Ca2+-calmodulin-dependent enzyme myosin light chain kinase (MLCK) that phosphorylates the regulatory light chain of myosin. Phosphorylation is a prerequisite for actin activation of myosin ATPase and contraction. It has been proposed that MLCK contains an inhibitory region that is regulated by calmodulin-binding. MLCK also contains a catalytic region and an actin-binding region. The function of the carboxy-terminus (approximately 24 kDa) is unknown, but preliminary evidence suggests that this portion is expressed independent of MLCK. The isolation of a partial cDNA for this enzyme makes it possible to use molecular biology techniques to further knowledge in this area by defining the relationship between the structure of these domains and function. This cDNA is 60% complete and includes the carboxy terminus, but the sequence of the amino terminal end of the molecule is unknown. The specific aims, proposed are: 1) Establish a bacterial system for the expression of active and Ca2+-calmodulin-dependent enzyme using the partial cDNA; 2) Define the domains contained within the partial cDNA using site-directed and deletion mutagenesis; 3) Determine the full-length sequence for MLCK by isolation of cDNA clones that extend the 5'-end of the partial cDNA; and 4) Characterize a new acidic protein (24 kDa) that has been isolated from smooth muscle and is thought to be identical with the carboxy-terminus of MLCK. MLCK is a key regulatory component in smooth muscle and a clear understanding of its mechanism is vital to our appreciation of normal smooth muscle function. This is a prerequisite for treatment of abnormal smooth muscle behavior; an important example is vascular smooth muscle. These studies will help in the design of pharmacological agents for the treatment of abnormal function.