ROLES OF MYOFILAMENT BOUND CALCIUM IN MUSCLE CONTRACTION

这项研究的总体长期目标是了解 肌浆游离钙升高调节的机制 脊椎动物横纹肌的收缩，特别是探索 钙离子与肌丝结合的方式可以调节和 受收缩事件的影响。要检验的假设提出了 钙与细丝的结合通过附着物得到增强 心肌和骨骼肌中肌动蛋白的自行车交叉桥。这 提出的现象已被引用为弗兰克的分子基础- Starling关系，并可能是收缩的其他特征的基础 横纹肌中的事件。这一假设将通过将 用定量电子方法研究钙沿肌节的分布 冻干样品的探针X射线微区分析及元素成像 甘油化兔腰肌冰冻切片，青蛙半腱肌病， 以及兔和青蛙的脑室小梁。纤维会被冻结在 含或不含镁三磷酸腺苷的溶液，以及不同水平的游离钙。 部分提取TNC和肌球蛋白调节蛋白后纤维的EPXMA 轻链将被用来确定任何观察到的 重叠区的钙结合增强。奥托伐酯或N- 苯基马来酰亚胺将被用来确定 依恋导致了这种现象。任何的空间范围 将进行钙化增强检查，以确定是否瘦 就钙结合而言，细丝表现为单个单位，或者 其他预测合作范围更短的模型是否 很受欢迎。还需要检查的是，在 钙离子沿细丝的亲和力，与相互作用无关 肌动蛋白和肌球蛋白之间。这些研究将为我们提供对 调节骨骼和骨骼中力的激活的基本机制 心肌。

The overall long term goal of this research is to understand the mechanisms by which elevation in myoplasmic free calcium regulates contraction of vertebrate striated muscle and in particular, to explore the ways in which calcium binding to myofilaments may modulate and be modulated by the contractile event. The hypothesis to be examined proposes that calcium binding to the thin filament is enhanced by the attachment of cycling crossbridges to actin in both cardiac and skeletal muscle. This proposed phenomenon has been cited as a molecular basis for the Frank- Starling relation, and may underlie other features of the contractile event in striated muscle. This hypothesis will be tested by mapping the distribution of calcium along the sarcomere using quantitative electron probe X-ray microanalysis (EPXMA) and elemental imaging of freeze dried cryosections of glycerinated rabbit psoas muscles, frog semitendinosis, and rabbit and frog ventricular trabeculae. Fibers will be frozen in solutions with or without MgATP and with varying levels of free calcium. EPXMA of fibers following partial extractions of TnC and myosin regulatory light chains would be used to identify the source of any observed enhancement in calcium binding in the overlap region. Orthovanidate or N- phenylmaleimide will be used to determine characteristics of the attachments with lead to this phenomenon. The spatial extent of any calcium enhancement will be examined to determine whether the thin filament behaves as a single unit with respect to calcium binding, or whether other models predicting a shorter range of cooperativity are favored. Also to be examined is whether there are intrinsic variations in calcium affinity along the thin filament, independent of interactions between actin and myosin. These studies will provide insight into the fundamental mechanisms which modulate activation of force in skeletal and cardiac muscle.