Levy H M, Moy W W
Biochim Biophys Acta. 1981 Apr 14;658(2):308-17. doi: 10.1016/0005-2744(81)90301-6.
The hydrolysis of MgATP by actomyosin gel at low ionic strength is known to show two unusual features: (1) an Arrhenius plot with a shallow slope in the higher temperature range (35-16 degrees C) and a steep slope in the lower temperature range (16-0 degrees C); (2) a rate curve of hydrolysis that begins with a 'burst' and falls to a lower steady-state level. Both of these can now be interpreted in terms of a specific, relatively slow transformation in the gel (t 1/2 = 9 s at 25 degrees C), induced by the binding of MgATP to the active sites of the myosin filaments. In the rate curves, this transformation is reflected in the transition from the burst rate (catalyzed by the original gel) to the steady-state rate (catalyzed by the modified gel). Importantly, this transition does not occur to a significant extent at low temperatures. Thus, in the typical nonlinear Arrhenius plot, where steady-state rates are used, the shallow slope in the high temperature range is a property of the modified gel, whereas the steep slope at low temperatures is a property of the original gel. Consistent with this interpretation, when the burst rates (presumably due to the original gel) were used in the high temperature range (and when substrate inhibition of hydrolysis by high levels of MgATP was avoided), the Arrhenius plot was linear over the entire temperature range (40-0 degrees C); the steep slope of this plot gives a high apparent heat of activation (25-30 kcal), similar to that reported for actin-activated hydrolysis by the soluble subfragment, heavy meromyosin. It is the steady-state form of the gel at high temperatures that gives a low apparent heat of activation (6-10 kcal). It was found that the regulatory proteins with calcium activate hydrolysis by the original form but have no effect on the steady-state form of the gel. Oxygen exchange measurements made during the burst and steady state at 25 degrees C indicate that the mechanism of hydrolysis is essentially the same for both, but that there is a higher effective actin concentration around the myosin sites in the original form.
已知在低离子强度下，肌动球蛋白凝胶对MgATP的水解呈现出两个不同寻常的特征：（1）在较高温度范围（35 - 16℃）内，阿累尼乌斯曲线的斜率较浅，而在较低温度范围（16 - 0℃）内斜率较陡；（2）水解速率曲线起始于一个“爆发期”，随后下降至较低的稳态水平。现在可以根据MgATP与肌球蛋白丝活性位点结合所诱导的凝胶中特定的、相对缓慢的转变（25℃时t1/2 = 9秒）来解释这两个现象。在速率曲线中，这种转变反映在从爆发速率（由原始凝胶催化）到稳态速率（由改性凝胶催化）的转变上。重要的是，这种转变在低温下不会显著发生。因此，在使用稳态速率的典型非线性阿累尼乌斯曲线中，高温范围内较浅的斜率是改性凝胶的特性，而低温下较陡的斜率是原始凝胶的特性。与此解释一致的是，当在高温范围内使用爆发速率（可能归因于原始凝胶）时（并且避免了高浓度MgATP对水解的底物抑制），阿累尼乌斯曲线在整个温度范围（40 - 0℃）内呈线性；该曲线较陡的斜率给出了较高的表观活化热（25 - 30千卡），类似于可溶性亚片段重酶解肌球蛋白对肌动蛋白激活水解所报道的值。正是高温下凝胶的稳态形式给出了较低的表观活化热（6 - 10千卡）。研究发现，含钙的调节蛋白可激活原始形式的水解，但对凝胶的稳态形式没有影响。在25℃的爆发期和稳态期进行的氧交换测量表明，两者的水解机制基本相同，但原始形式的肌球蛋白位点周围有效肌动蛋白浓度更高。
