CALCIUM AND CONTRACTILE ACTIVATION IN STRIATED MUSCLE

拟议工作的目的是调查 化学机械转导(即化学与机械的比率 活动)，由横纹肌的收缩装置在各种情况下 实验条件。这将通过同时测量 细丝结皮纤维中的等长力与ATP水解力 调节肌肉(来自青蛙和兔子)以及来自粗丝 调节肌肉(来自扇贝)。ATPase将使用 连接酶分析，通过观察NADH荧光的减少 这个实验室已经建造了微型荧光仪。在……里面 具体地说，目的是调查紧张成本(即 ATPase/FORCE的比值)随激活钙的水平而变化。 兔Will快、慢收缩纤维的张力成本比较 指出纤维类型之间的可能差异，同时比较 来自兔子和青蛙的快速抽动纤维的张力-成本将进行调查 可能的物种差异。张力-成本的进一步比较 前述细丝调节的肌肉与 粗丝调节扇贝肌肉将评估 对收缩经济的监管模式。进一步的实验将 在已知影响因素的条件下调查紧张-成本 收缩机械的钙敏感性和/或力产生， 即：溶液pH值、底物(镁三磷酸腺苷)、离子强度、镁离子、 温度、底物/产物比(即自由能)，以及 用氧化氢(D20)取代溶剂水的同位素。一个 一种机械长度控制装置，可快速扰动长度 蒙皮的纤维段将被用来监测纤维的硬度作为一种测量 连接的交叉桥的数量。在快速释放之后， 将研究武力恢复的多阶段过程，以指示变化 在横桥旋转和脱离的速度上。最后，最大的 缩短速度将使用松弛测试技术进行测量。在……里面 结合FORCE和ATPase，这些交叉桥数和 行为将提供对这些不同的 实验条件对收缩装置有影响。这项工作将 也具有临床意义，因为观察到的负离子营养不良 骨骼肌和心肌的疲劳和缺氧与 细胞内pH、底物和自由能的变化。目前， 然而，目前尚不清楚这些变化如何影响收缩 进程。希望这项研究能对这一点有所帮助。 有问题。

The aim of the proposed work is to investigate the economy of chemomechanical transduction (i.e. the ratio of chemical to mechanical activity) by the contractile apparatus of striated muscle under a variety of experimental conditions. This will be done by simultaneously measuring isometric force and ATP hydrolysis in skinned fibers from thin-filament regulated muscle (from frog and rabbit) as well as from thick-filament regulated muscle (from scallop). ATPase will be estimated using a linked-enzyme assay, by observing the decrease of NADH fluorescence using a microfluorometer which has been constructed in this laboratory. In specific, the aim is to investigate whether the tension-cost (i.e. the ratio of ATPase/force) varies with the level of activating calcium. Comparison of tension-cost of fast and slow twitch fibers from rabbit will indicate likely differences between fiber types, while comparison of tension-cost of fast twitch fibers from rabbit and frog will investigate possible species differences. Further comparison of tension-cost of the aforementioned thin-filament regulated muscles with that of a thick-filament regulated scallop muscle will assess the influence of regulatory mode on contractile economy. Further experiments will investigate tension-cost under conditions which are known to influence calcium sensitivity and/or force production of the contractile machinery, to wit: variations of solution pH, substrate (MgATP), ionic strength, Mg2+, temperature, and substrate/product ratios (i.e. free energy), as well as isotopic substitution of solvent water by deuterium oxide (D20). A mechanical length control apparatus which can rapidly perturb the length of skinned fiber segments will be used to monitor fiber stiffness as a measure of the number of attached crossbridges. After a quick release, the multiphasic process of force recovery will be studied to indicate changes in the rate of crossbridge rotation and detachment. Lastly, the maximal velocity of shortening will be measured using a slack-test technique. In conjunction with force and ATPase, these measures of crossbridge number and behavior will provide insight into the mechanisms by which these various experimental conditions affect the contractile apparatus. This work will be of clinical interest, as well, in that the negative ionotrophy observed with fatigue and hypoxia of skeletal and cardiac muscles is associated with changes in intracellular pH, substrate, and free energy. At present, however, it is not clear how these changes influence the contractile process. It is hoped that the present study might shed some light on this problem.