CALCIUM & CONTRACTILE ACTIVATION IN STRIATED MUSCLE

拟议工作的目的是调查的过程中， 横纹肌收缩器的化学机械传递 肌肉. 转换的经济性（即“紧张-成本”， ATP酶/力）将通过同时测量等长力进行研究 和ATP水解的化学皮肤纤维从快速和缓慢- 抽搐骨骼肌 ATP酶将用连接的酶来估计 使用专门设计的显微荧光计测定。 此外该 最大无负荷缩短速度（Vmax）、刚度和 在短暂的轻负荷等渗后， 将测量缩短。 具体而言，目的是调查 激活Ca ~（2+）水平不同，张力-代价也不同。 比较快缩和慢缩肌纤维的张力-成本， 表明纤维类型之间可能存在差异。 肌钙蛋白C的作用 (TnC)和肌球蛋白磷酸化轻链（P-LC） 将通过交换TnC或P-LC来研究差异。 的影响 还将研究P-LC的磷酸化。 纤维型和TnC或P-LC 将使用超灵敏聚丙烯酰胺凝胶 单纤维电泳。 除了Ca 2+，我们还将关注 主要是pH值、无机磷酸盐（Pi）和 ADP，因为已知这些变化对 跨桥循环的重要步骤。 此外，他们被认为 在观察到的收缩力和Vmax下降中起主要作用， 完整肌肉的疲劳。 在这方面，pH和Pi尤其重要。 重要;因此它们对收缩器官的相互作用 将使用我们将合成的Pi的化学类似物进行检查。 在 结合等长收缩力和ATP酶等力学指标 跨桥的数量和行为将提供详细的洞察力， 疲劳或缺氧不利地影响心肌收缩的机制 过程

The aim of the proposed work is to investigate the process of chemomechanical transduction by the contractile apparatus of striated muscle. The economy of transduction (i.e. "tension-cost", the ratio of ATPase/force) will be studied by simultaneously measuring isometric force and ATP hydrolysis in chemically skinned fibers from both fast- and slow- twitch skeletal muscle. ATPase will be estimated with a linked enzyme assay using a specially-designed microfluorometer. In addition, the maximal velocity of unloaded shortening (Vmax), stiffness, and the rate of redevelopment of force after a brief period of lightly-loaded isotonic shortening will be measured. In specific, the aim is to investigate whether the tension-cost varies with the level of activating Ca2+. Comparison of tension-cost of fasts-twitch and slow-twitch fibers will indicate likely differences between fiber types. The role of troponin C (TnC) and myosin phosphorylatable light chains (P-LC) in any fiber-type differences will be studied by exchange of TnC or P-LCs. The effect of phosphorylation of P-LCs will also be studied. Fiber-type and TnC or P-LC status will be verified using ultrasensitive polyacrylamide gel electrophoresis of single fibers. In addition to Ca2+, we will focus primarily on the effects of changes in pH, inorganic phosphate (Pi), and ADP because these variations are known to have significant effects on important steps of the cross-bridge cycle. Moreover, they are thought to play major roles in the decline of contractile force and Vmax observed with fatigue of intact muscle. In this regard, pH and Pi are especially important; therefore their interacting effects on the contractile apparatus will be examined using chemical analogs of Pi which we will synthesize. In conjunction with isometric force and ATPase, the other mechanical measures of cross-bridge number and behavior will provide detailed insight into the mechanisms by which fatigue or hypoxia adversely affect the contractile process.