STRUCTURE VS. FUNCTION IN MYOSIN LIGHT CHAIN KINASE

结构对比

• 批准号: 3362368
• 负责人: Vince Guerriero
• 金额: $ 11.16万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3362368
• 关键词: actins; adenosinetriphosphatase; calmodulin; carboxyl group; cardiovascular pharmacology; chemical structure function; complementary DNA; drug design /synthesis /production; enzyme induction /repression; hormone regulation /control mechanism; messenger RNA; muscle contraction; myosin light chain kinase; myosins; phosphorylation; phosphotransferases; site directed mutagenesis; smooth muscle; vascular smooth muscle nervous control

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.

该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。监管 平滑肌收缩是由 Ca2+-钙调蛋白依赖性酶肌球蛋白控制的 轻链激酶 (MLCK)，磷酸化调节轻链 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 受钙调蛋白结合调节的抑制区域。 MLCK也 包含催化区和肌动蛋白结合区。的功能 羧基末端（约 24 kDa）未知，但初步确定 有证据表明该部分的表达与 MLCK 无关。这 分离该酶的部分 cDNA 使得可以使用 分子生物学技术通过定义进一步加深该领域的知识 这些域的结构和功能之间的关系。这 cDNA 60% 完整，包含羧基末端，但序列 该分子的氨基末端未知。具体目标， 建议：1）建立表达活性物质的细菌系统 和使用部分cDNA的Ca2+-钙调蛋白依赖性酶； 2) 定义 使用定点和删除来确定部分 cDNA 中包含的结构域 诱变； 3) 通过分离确定MLCK的全长序列 延伸部分 cDNA 5' 端的 cDNA 克隆； 4) 表征 从平滑肌中分离出一种新的酸性蛋白 (24 kDa) 被认为与 MLCK 的羧基末端相同。 MLCK 是平滑肌的关键调节成分，具有明确的作用 了解其机制对于我们理解正常现象至关重要 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。