Diffee G M, Greaser M L, Reinach F C, Moss R L
Department of Physiology, University of Wisconsin, Madison 53706, USA.
Biophys J. 1995 Apr;68(4):1443-52. doi: 10.1016/S0006-3495(95)80317-6.
Each myosin molecule contains two heavy chains and a total of four low-molecular weight light chain subunits, two "essential" and two "regulatory" light chains (RLCs). Although the roles of myosin light chains in vertebrate striated muscle are poorly understood at present, recent studies on the RLC have suggested that it has a modulatory role with respect to Ca2+ sensitivity of tension and the rate of tension development, effects that may be mediated by Ca2+ binding to the RLC. To examine possible roles of the RLC Ca2+/Mg2+ binding site in tension development by skeletal muscle, we replaced endogenous RLC in rabbit skinned psoas fibers with an avian mutant RLC (D47A) having much reduced affinity for divalent cations. After replacement of up to 80% of the endogenous RLC with D47A RLC, maximum tension (at pCa 4.5) was significantly reduced compared with preexchange tension, and the amount of decrease was directly related to the extent of D47A exchange. Fiber stiffness changed in proportion to tension, indicating that the decrease in tension was due to a decrease in the number of tension-generating cross-bridges. Decreases in both tension and stiffness were substantially, although incompletely, reversed after reexchange of native RLC for D47A. RLC exchange was also performed using a wild-type RLC. Although a small decrease in tension was observed after wild-type RLC exchange, the decrease was not proportional to the extent of RLC exchange and was not reversed by reexchange of the native RLC. D47A exchange also decreased the Ca2+ sensitivity of tension and reduced the apparent cooperativity of tension development. The results suggest that divalent cation binding to myosin RLC plays an important role in tension generation in skeletal muscle fibers.
每个肌球蛋白分子包含两条重链和总共四个低分子量轻链亚基，即两条“必需”轻链和两条“调节”轻链（RLC）。尽管目前对脊椎动物横纹肌中肌球蛋白轻链的作用了解甚少，但最近对RLC的研究表明，它对张力的Ca2 +敏感性和张力发展速率具有调节作用，这些作用可能是由Ca2 +与RLC结合介导的。为了研究RLC Ca2 + / Mg2 +结合位点在骨骼肌张力发展中的可能作用，我们用对二价阳离子亲和力大大降低的禽类突变RLC（D47A）替代了兔去皮肤腰大肌纤维中的内源性RLC。用D47A RLC替代高达80％的内源性RLC后，与交换前的张力相比，最大张力（在pCa 4.5时）显着降低，并且降低的量与D47A交换的程度直接相关。纤维刚度与张力成比例变化，表明张力降低是由于产生张力的横桥数量减少。在用天然RLC重新交换D47A后，张力和刚度的降低虽然不完全但基本上得到了逆转。也使用野生型RLC进行RLC交换。尽管在野生型RLC交换后观察到张力有小幅下降，但下降与RLC交换的程度不成比例，并且不能通过天然RLC的重新交换来逆转。D47A交换还降低了张力的Ca2 +敏感性并降低了张力发展的表观协同性。结果表明，二价阳离子与肌球蛋白RLC的结合在骨骼肌纤维的张力产生中起重要作用。
