BIOCHEMISTRY OF CONTRACTILE PROTEINS

收缩蛋白的生物化学

• 批准号: 3337322
• 负责人: David John Hartshorne
• 金额: $ 23.02万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-07-01 至 1988-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3337322
• 关键词: adenosinetriphosphatase; biological information processing; calcium; crosslink; enzyme structure; fluorescence spectrometry; immunofluorescence technique; magnesium; muscle contraction; muscle proteins; nuclear magnetic resonance spectroscopy; phosphorylation; radiotracer; smooth muscle; striated muscles; vascular smooth muscle

Our long range goal is to gain a better understanding of the regulatory mechanisms in smooth muscle. Smooth muscle is essential for normal body function and is the contractile element of arteries, veins, intestines, uterus, etc. It is known that an increase in the intracellular concentration of CA++ induces contraction of smooth muscle. The mechanism by which the changes in CA++ concentration are detected by the contractile apparatus and then processed to result in either contraction or relaxation is still controversial. There are two basic theories: the most popular hypothesis is that phosphorylation of myosin by a myosin light chain kinase (MLCK) activates the contractile apparatus and leads to contraction, deactivation (relaxation) is achieved by dephosphorylation of myosin by a myosin light chain phophatase (MLCP); the alternative theory is that regulation is achieved by a mechanism termed leiotonin and does not involve myosin phosphorylation. There is considerable evidence to indicate that the phosphorylation theory forms at least part of the regulatory mechanism and the focus of this proposal is to examine in detail different aspects of this theory. The association of the MLCK with the contractile elements will be studied. Evidence suggests that MLCK binds to actin. This will be confirmed and the conditions of binding established. If the MLCK is restricted to thin filaments the extent of filament overlap will dictate the level of phosphorylation. Improved procedures for the isolation of MLCK will be investigated. There are some data to sugget that all previously isolated MLCKs may be derived from a larger precursor. The phosphorylation reaction will also be analyzed in more detail since it is possible that phosphorylation is sequential rather than random, and whether this is a general property of myosin molecules will be tested by studying phosphorylation of smooth muscle and skeletal muscle muscle myosins and their subfragments. An important objective is to estgablish the role of phosphorylation and several experiments are suggested in which phosphorylation can be correlated to ATPase activity of actomyosin or superprecipitation. Whether or not non-cycling myosin-actin attachments are formed under in vitro conditions will be investigated. The data from the above experiments will be evaluated to assess the role of myosin phosphorylation and to question whether additional mechanisms are indicated. The latter will also be tested using CA++ insensitive MLCK.

我们的长期目标是更好地了解监管 平滑肌中的机制。 平滑肌对于正常身体至关重要 功能，是动脉、静脉、肠的收缩元件， 子宫等。已知细胞内的增加 CA++浓度诱导平滑肌收缩。 机制 CA++浓度的变化通过收缩检测 设备然后进行处理以导致收缩或松弛 仍有争议。 有两种基本理论：最流行的 假设是肌球蛋白轻链激酶磷酸化肌球蛋白 (MLCK) 激活收缩装置并导致收缩， 失活（松弛）是通过肌球蛋白的去磷酸化来实现的 肌球蛋白轻链磷酸酶（MLCP）；另一种理论是 调节是通过一种称为“leiotonin”的机制实现的，不涉及 肌球蛋白磷酸化。 有大量证据表明 磷酸化理论至少构成调节机制的一部分 该提案的重点是详细审查 这个理论。 MLCK 与收缩元件的关联 将被研究。 有证据表明 MLCK 与肌动蛋白结合。 这将是 确认并确定绑定条件。 如果 MLCK 是 仅限于细丝，丝重叠的程度将决定 磷酸化水平。 改进隔离程序 MLCK将被调查。 有一些数据表明，所有 先前分离的 MLCK 可能源自较大的前体。 这 磷酸化反应也将被更详细地分析，因为它是 磷酸化可能是连续的而不是随机的，以及是否 这是肌球蛋白分子的一般特性，将通过研究进行测试 平滑肌和骨骼肌肌球蛋白的磷酸化和 他们的子片段。 一个重要目标是确立 磷酸化和一些实验被建议，其中 磷酸化可能与肌动球蛋白或肌动球蛋白的 ATP 酶活性相关 超降水。 是否有非循环肌球蛋白-肌动蛋白附着 将研究在体外条件下形成的。 数据来自 将评估上述实验以评估肌球蛋白的作用 磷酸化并质疑是否有其他机制 表明的。 后者也将使用 CA++ 不敏感 MLCK 进行测试。