Ribeiro A S, Salerno V P, Sorenson M
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590 RJ, Brazil.
Biochim Biophys Acta. 2005 May 15;1748(2):165-73. doi: 10.1016/j.bbapap.2004.12.016. Epub 2005 Jan 19.
Access to different intermediates that follow ATP cleavage in the catalytic cycle of skeletal muscle actomyosin is a major goal of studies that aim toward an understanding of chemomechanical coupling in muscle contraction. 2,4-Dinitrophenol (DNP, 10(-2) M) inhibits muscle contraction, even though it accelerates the ATPase activity of isolated myosin. Here we used myosin subfragment 1 (S1), acto-S1 and mammalian skinned fibers to investigate the action of DNP in the presence of actin. DNP increases acto-S1 affinity and at the same time reduces the maximum rate of turnover as [actin]-->infinity. In skinned fibers, isometric force is reduced to the same extent (K0.5 approximately equal to 6 mM). Although actin activates Pi release from S1 at all DNP concentrations tested, the combination of enhanced S1 activity and reduced acto-S1 activity leads to a reduction in the ratio of these two rates by a factor of 30 at the highest DNP concentration tested. This effect is seen at low as well as at high actin concentrations and is less pronounced with the analog meta-nitrophenol (MNP), which does not inhibit the acto-S1 ATPase. Arrhenius plots for acto-S1 are parallel and linear between 5 and 30 degrees C, indicating no abrupt shifts in rate-limiting step with either DNP or MNP. Analysis of the reduction in isometric force with increasing Pi concentrations suggests that DNP and MNP stabilize weakly bound cross-bridges (AM.ADP.Pi). In addition, MNP (10(-2) M) increases the apparent affinity for Pi.
在骨骼肌肌动球蛋白催化循环中，获取ATP水解后不同的中间体，是旨在理解肌肉收缩中化学机械偶联研究的主要目标。2,4 -二硝基苯酚（DNP，10⁻² M）抑制肌肉收缩，尽管它能加速分离的肌球蛋白的ATP酶活性。在此，我们使用肌球蛋白亚片段1（S1）、肌动蛋白 - S1和哺乳动物的去膜纤维来研究在肌动蛋白存在下DNP的作用。随着肌动蛋白浓度趋近于无穷大，DNP增加了肌动蛋白 - S1的亲和力，同时降低了最大周转速率。在去膜纤维中，等长力降低到相同程度（K0.5约等于6 mM）。尽管在所有测试的DNP浓度下肌动蛋白都能激活S1释放无机磷酸（Pi），但在最高测试DNP浓度下，增强的S1活性和降低的肌动蛋白 - S1活性共同作用，导致这两个速率的比值降低了30倍。在低和高肌动蛋白浓度下均可见此效应，而对不抑制肌动蛋白 - S1 ATP酶的类似物间硝基苯酚（MNP）而言，这种效应不太明显。肌动蛋白 - S1的阿累尼乌斯曲线在5至30摄氏度之间是平行且线性的，表明使用DNP或MNP时限速步骤均无突然变化。随着Pi浓度增加对等长力降低的分析表明，DNP和MNP稳定了弱结合的横桥（AM.ADP.Pi）。此外，MNP（10⁻² M）增加了对Pi的表观亲和力。
