MECHANISM OF CARDIAC ACTOMYOSIN NTP HYDROLYSIS

描述（改编自申请人的摘要）：本申请的主要目的 我们的工作是扩展我们对 三磷酸核苷（NTP）水解的机理和机械性质 肌肉纤维的性质，重点是确定哪一步或 核苷酸三磷酸水解机制的步骤限制了 缩短速度 研究人员已经证明，一系列核苷 三磷酸盐（NTPS）在支持起酥油的能力方面差异很大 和运动性，如在皮肤肌肉纤维中测量的（与 Drs. Roger Cooke和艾德·佩特）并使用“体外”运动性测定。 在 在这项提案的头两年，调查人员计划完成 系列核苷三磷酸水解机理的研究 通过肌动球蛋白使用停流荧光，光散射和快速 化学淬火法 通过这项工作，他们计划确定 水解机制的步骤速率常数的变化改变了 肌肉的机械和运动特性。 为了做到这一点， 研究人员将详细比较 肌动球蛋白NTP水解的动力学机制 肌纤维的机械性能和缩短速度， 体外运动性测定。 一个平行的方法是确定氨基酸序列的差异 这些细胞负责酶和收缩的差异， 不同肌球蛋白的活性。 在氨基酸上有7%的差异 α和β心肌肌球蛋白之间的酸性序列， 四个簇负责机械和酶的差异， 心肌肌球蛋白同工酶的性质。 一个类似的模式， 序列的改变会导致肌动蛋白 激活的ATP水解，缩短速度的五倍差异， 之间ADP与acto-S1解离的速率相差十倍 心肌和快速骨骼肌球蛋白。 最近的数据（Uyeda等人，1994年）表明， 肌动蛋白激活ATP水解的速率取决于 氨基酸624-638的序列，其位于 肌球蛋白分子的25和50 kDA区域。 研究人员计划使用 嵌合肌球蛋白（嵌合酶）的重组表达的组合， 稳态动力学、稳态前动力学和运动性测定 测量以确定哪些氨基酸变化是导致 观察到的动力学机制和功能差异的差异。 了解哪些氨基酸会改变代谢速率和平衡常数 肌肉中水解机制和缩短速度将提供 更好地理解肌肉收缩的分子机制。

DESCRIPTION (Adapted from the applicant's abstract): A primary aim of this work is to extend our understanding of the relationship between the mechanism of nucleoside triphosphate (NTP) hydrolysis and the mechanical properties of muscle fibers with an emphasis upon determining which step or steps of the nucleotide triphosphate hydrolysis mechanism limits the maximum shortening velocity. Investigators have shown that a series of nucleoside triphosphates (NTPS) differ widely in their ability to support shortening and motility as measured in skinned muscle fibers (in collaboration with Drs. Roger Cooke and Ed Pate) and using the `in vitro' motility assay. In the first two years of this proposal the investigators plan to complete the study of the mechanism of hydrolysis of a series of nucleoside triphosphates by actomyosin using stopped-flow fluorescence, light scattering, and rapid chemical quench method. From this work they plan to determine for which steps of the hydrolysis mechanism changes in rate constants alter the mechanical and motile properties of muscle. To accomplish this the investigators will make a detailed comparison of the rate constants of the kinetic mechanism of the actomyosin NTP hydrolysis mechanism with the mechanical properties of muscle fibers and shortening velocity measured with the in vitro motility assay. A parallel approach is to determine the differences in amino acid sequence that are responsible for the differences in enzymatic and contractile activities of different myosins. A seven percent difference in the amino acid sequence between alpha and beta cardiac myosin primarily grouped in four clusters is responsible for the difference in mechanical and enzymatic properties of cardiac myosin isozymes. A similar pattern of more extensive sequence changes produces a three fold difference in the rates of actin activated ATP hydrolysis, a five fold difference in shortening velocity, and a ten fold difference in rate of ADP dissociation from acto-S1 between cardiac and fast skeletal myosin. Recent data (Uyeda et al. 1994) indicate that the rate of actin-activated ATP hydrolysis is dependent upon the sequence of amino acids 624-638, which are located in the junction between the 25 and 50 kDA regions of the myosin molecule. Investigators plan to use a combination of recombinant expression of chimeric myosins (chimerzymes), steady state kinetics, pre-steady state kinetics, and motility assay measurements to determine which amino acid changes are responsible for the observed differences in the kinetic mechanism and functional differences. Understanding which amino acids alter the rate and equilibrium constants of the hydrolysis mechanism and shortening velocity in muscle will provide a better understanding of the molecular mechanism of muscle contraction.