From the Department of Biochemistry, University of Nouakchott, Nouakchott 5026, Mauritania.
Department of Biochemistry, Memorial University, St. Johns, Newfoundland A1B 3X9, Canada.
J Biol Chem. 2010 Oct 22;285(43):32760-32769. doi: 10.1074/jbc.M109.098228. Epub 2010 Aug 9.
We have studied the mechanism of activation of native cardiac thin filaments by calcium and rigor myosin. The acceleration of the rate of 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) dissociation from cardiac myosin-S1-mdADP-P(i) and cardiac myosin-S1-mdADP by native cardiac muscle thin filaments was measured using double mixing stopped-flow fluorescence. Relative to inhibited thin filaments (no bound calcium or rigor S1), fully activated thin filaments (with both calcium and rigor-S1 bound) increase the rate of product dissociation from the physiologically important pre-power stroke myosin-mdADP-P(i) by a factor of ∼75. This can be compared with only an ∼6-fold increase in the rate of nucleotide diphosphate dissociation from nonphysiological myosin-mdADP by the fully activated thin filaments relative to the fully inhibited thin filaments. These results show that physiological levels of regulation are not only dependent on the state of the thin filament but also on the conformation of the myosin. Less than 2-fold regulation is due to a change in affinity of myosin-ADP-P(i) for thin filaments such as would be expected by a simple "steric blocking" of the myosin-binding site of the thin filament by tropomyosin. Although maximal activation requires both calcium and rigor myosin-S1 bound to the cardiac filament, association with a single ligand produces ∼70% maximal activation. This can be contrasted with skeletal thin filaments in which calcium alone only activated the rate of product dissociation ∼20% of maximum, and rigor myosin produces ∼30% maximal activation.
我们研究了钙离子和僵硬肌球蛋白激活天然心肌细肌丝的机制。使用双混合停流荧光法测量了天然心肌细肌丝对心肌肌球蛋白 S1-mdADP-P(i)和心肌肌球蛋白 S1-mdADP 的 2'-脱氧-3'-O-(N-甲基邻氨基苯甲酰基)ADP(mdADP)的解离速率的加速作用。与抑制性细肌丝（无结合钙或僵硬 S1）相比，完全激活的细肌丝（同时结合钙和僵硬 S1）使生理上重要的预功构象肌球蛋白-mdADP-P(i)的产物解离速率增加了约 75 倍。这可以与完全激活的细肌丝相对于完全抑制的细肌丝使非生理肌球蛋白-mdADP 的核苷酸二磷酸解离速率仅增加约 6 倍进行比较。这些结果表明，生理调节水平不仅取决于细肌丝的状态，还取决于肌球蛋白的构象。由于肌球蛋白与肌动蛋白结合的亲和力发生变化，如肌球蛋白结合位点被肌球蛋白抑制素的“空间位阻”所阻止，小于 2 倍的调节是由于肌球蛋白-ADP-P(i)与细肌丝的亲和力变化所致。虽然钙和僵硬肌球蛋白 S1 结合到心肌丝上都需要最大程度的激活，但与单个配体结合可产生约 70%的最大激活。这与骨骼肌细肌丝形成对比，在骨骼肌细肌丝中，钙单独仅激活产物解离的速率约为最大的 20%，而僵硬肌球蛋白产生约 30%的最大激活。
