CALCIUM & CONTRACTILE ACTIVATION IN STRIATED MUSCLE

拟议工作的目的是调查 纹状肌收缩器的化学机械转导 肌肉。转导的经济性(即“张力-成本”、 ATPase/force)将通过同时测量等长力来研究 以及来自快速和慢速皮肤的化学剥离纤维中的ATP水解酶 抽动骨骼肌。ATPase将用连接的酶来估计 使用专门设计的微型荧光计进行化验。此外， 最大无负荷缩短率(Vmax)、最大刚度和最大下降率 在短暂的轻负荷等张运动后力量的重新发展 将测量做空。具体地说，目的是调查 张力-成本是否随激活钙离子水平的不同而变化。 快抽与慢抽纤维的张力成本比较 指出不同纤维类型之间的可能差异。肌钙蛋白C的作用 (TNC)和肌球蛋白可磷酸化轻链(P-LC)在任何纤维类型中 将通过交换TNC或P-LC来研究差异。的影响 P-LCS的磷酸化也将被研究。光纤类型和TNC或P-LC 状态将使用超敏感的聚丙烯酰胺凝胶进行验证 单纤维的电泳法。除了钙离子，我们还将关注 主要取决于pH值、无机磷(PI)和 ADP，因为已知这些变异对 这是跨桥循环的重要步骤。此外，他们被认为是 在收缩力和Vmax的下降中起主要作用。 完整肌肉的疲劳。在这方面，pH和等电点尤其重要 很重要；因此，它们对收缩装置的相互作用 将使用我们将合成的PI的化学类似物进行检测。在……里面 结合等长力和ATPase，其他机械措施 将提供对跨桥数量和行为的详细了解 疲劳或缺氧对心肌收缩产生不利影响的机制 进程。

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.