Mulhern S A, Eisenberg E
Biochemistry. 1976 Dec 28;15(26):5702-8. doi: 10.1021/bi00671a004.
It has been postulated that, during the hydrolysis of ATP, both normal and SH1-blocked heavy meromyosin undergo a rate-limiting transition from a refractory state which cannot bind to actin to a nonrefractory state which can bind to actin. This model leads to several predictions which were studied in the present work. First, the fraction of heavy meromysin or subfragment 1 which remains unbound to actin when the ATPase equals Vmax should have the same properties as the original protein. In the present study it was determined that the unbound protein has normal ATPase activity which suggests that it is unbound to actin for a kinetic reason rather than because it is a permanently altered form of the myosin. Second, if the heavy meromyosin heads act independently half as much subfragment 1 as heavy meromyosin should bind to actin. Experiments in the ultracentrifuge demonstrate that about half as much subfragment 1 as heavy meromyosin sediments with the actin at Vmax. Third, the ATP turnover rate per actin monomer at infinite heavy meromyosin concentration should be much higher than the ATP turnover rate per heavy meromyosin head at infinite actin concentration. This was found to be the case for SH1-blocked heavy meromyosin since, even at very high concentrations of SH1-blocked heavy meromyosin, in the presence of a fixed actin concentration, the actin-activated ATPase rate remained proportional to the SH1-blocked heavy meromyosin concentration. All of these results tend to confirm the refractory state model for both SH1-blocked heavy meromyosin and unmodified heavy meromyosin and subfragment 1. However, the nature of the small amount of heavy meromyosin which does bind to actin in the presence of ATP at high actin concentration remains unclear.
据推测，在ATP水解过程中，正常的和SH1阻断的重酶解肌球蛋白都会经历一个限速转变，即从不能与肌动蛋白结合的不应期状态转变为可以与肌动蛋白结合的非不应期状态。该模型得出了几个预测，本研究对其进行了探讨。首先，当ATP酶活性等于Vmax时，未与肌动蛋白结合的重酶解肌球蛋白或亚片段1的部分应具有与原始蛋白质相同的特性。在本研究中，已确定未结合的蛋白质具有正常的ATP酶活性，这表明它未与肌动蛋白结合是出于动力学原因，而非因为它是肌球蛋白的永久改变形式。其次，如果重酶解肌球蛋白头部独立起作用，那么与肌动蛋白结合的亚片段1应只有重酶解肌球蛋白的一半。超速离心实验表明，在Vmax时，与肌动蛋白一起沉降的亚片段1约为重酶解肌球蛋白的一半。第三，在无限重酶解肌球蛋白浓度下，每个肌动蛋白单体的ATP周转速率应远高于在无限肌动蛋白浓度下每个重酶解肌球蛋白头部的ATP周转速率。对于SH1阻断的重酶解肌球蛋白来说，情况确实如此，因为即使在非常高的SH1阻断重酶解肌球蛋白浓度下，在固定肌动蛋白浓度存在的情况下，肌动蛋白激活的ATP酶速率仍与SH1阻断的重酶解肌球蛋白浓度成正比。所有这些结果都倾向于证实SH1阻断的重酶解肌球蛋白以及未修饰的重酶解肌球蛋白和亚片段1的不应期状态模型。然而，在高肌动蛋白浓度下存在ATP时，少量确实与肌动蛋白结合的重酶解肌球蛋白的性质仍不清楚。
