STRUCTURE VS. FUNCTION IN MYOSIN LIGHT CHAIN KINASE

这个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。规管 平滑肌收缩是通过钙依赖的钙调蛋白酶肌球蛋白实现的 轻链激酶(MLCK)，磷酸化调节轻链 肌球蛋白。肌球蛋白的磷酸化是肌动蛋白激活的先决条件 ATPase和收缩。有人提出，MLCK包含一个 受钙调蛋白结合调节的抑制区域。MLCK也 包含一个催化区和一个肌动蛋白结合区。的功能 羧基末端(约24 kDa)未知，但初步 有证据表明，这一部分的表达独立于MLCK。这个 这种酶的部分cdna的分离使其有可能用于 通过定义分子生物学技术来加深这一领域的知识 这些结构域的结构和功能之间的关系。这 CDNA60%完成，并包括羧基末端，但序列 该分子的氨基末端是未知的。具体的目标， 建议：1)建立活性基因表达的细菌系统 和钙调素依赖酶；2)定义 利用定点定位和缺失的方法获得部分cDNA中包含的结构域 突变；3)通过分离确定MLCK的全长序列 延伸部分cDNA5‘端的cdna克隆；以及4)特征 一种新的酸性蛋白(24 KDa)，已从平滑肌和 被认为与MLCK的羧基末端相同。 MLCK是平滑肌中的一个关键调节成分，是一种清晰的 对其机制的理解对于我们认识正常是至关重要的。 平滑的肌肉功能。这是治疗异常的先决条件 平滑肌肉的行为；一个重要的例子是血管平滑肌。 这些研究将有助于设计用于治疗癌症的药物。 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.