Diffee G M, Patel J R, Reinach F C, Greaser M L, Moss R L
Department of Physiology, University of Wisconsin, Madison 53706, USA.
Biophys J. 1996 Jul;71(1):341-50. doi: 10.1016/S0006-3495(96)79231-7.
We examined the kinetic properties of rabbit skinned skeletal muscle fibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutation within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the endogenous RLC was replaced by the mutant, maximum tension declined to approximately 60% of control and the rate constant of active tension redevelopment (ktr) after mechanical disruption of cross-bridges was reduced to approximately 70% of control. This reduction in ktr was not an indirect effect on kinetics due to a reduced number of strongly bound myosin heads, because when the strongly binding cross-bridge analog N-ethylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fibers, there was no effect upon maximum ktr. Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the number of strongly bound cross-bridges, suggesting that the force per cross-bridge was not significantly affected by the presence of D47A RLC. In contrast to the effects on ktr, the rate of tension relaxation in steadily activated fibers after flash photolysis of the Ca2+ chelator diazo-2 increased by nearly twofold after D47A exchange. We conclude that the incorporation of the nondivalent cation-binding mutant of myosin RLC decreases the proportion of cycling cross-bridges in a force-generating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest that divalent cation binding to myosin RLC plays an important role in modulating the kinetics of cross-bridge attachment and detachment.
我们研究了兔去表皮骨骼肌纤维的动力学特性，其中内源性肌球蛋白调节轻链（RLC）部分被突变型RLC（D47A）取代，该突变型在Ca2+/Mg2+结合位点含有一个点突变，严重降低了其对二价阳离子的亲和力。我们发现，当约50%的内源性RLC被突变体取代时，最大张力降至对照的约60%，并且在横桥机械破坏后主动张力重建的速率常数（ktr）降至对照的约70%。ktr的这种降低不是由于强结合肌球蛋白头部数量减少而对动力学产生的间接影响，因为当将强结合横桥类似物N-乙基马来酰亚胺修饰的肌球蛋白亚片段1（NEM-S1）添加到纤维中时，对最大ktr没有影响。D47A交换后纤维刚度下降，表明强结合横桥数量减少，这表明每个横桥的力不受D47A RLC存在的显著影响。与对ktr的影响相反，在Ca2+螯合剂重氮-2闪光光解后，稳定激活的纤维中张力松弛速率在D47A交换后增加了近两倍。我们得出结论，肌球蛋白RLC的非二价阳离子结合突变体的掺入通过降低产生力的桥的形成速率和增加脱离速率，降低了处于产生力状态的循环横桥的比例。这些结果表明，二价阳离子与肌球蛋白RLC的结合在调节横桥附着和脱离的动力学中起重要作用。
