Regulation Of Smooth and Nonmuscle Myosin

位于颈部的20 kDa调节轻链(RLC)的磷酸化调节了平滑肌和非肌肉肌球蛋白II的活性。该区域包含肌球蛋白重链、RLC和基本轻链(ELC)的单个α-螺旋片段。脊椎动物有三个编码非肌肉肌球蛋白II的基因，分别称为IIA和IIB。血小板仅表达IIA亚型，而脑主要表达IIB亚型。大多数其他细胞表达IIA和IIB的比例几乎相等，因为它们位于不同的位置。我们实验室以前的工作表明，非肌肉肌球蛋白IIA和肌球蛋白IIB具有不同的酶和运动特性。为了更好地了解这种差异的基础，我们设计了肌球蛋白IIA和肌球蛋白IIB S-1类片段用于杆状病毒表达。我们已经完全表征了这两个分子在肌动蛋白存在和不存在的情况下的瞬时酶动力学。这两种肌球蛋白都具有动力学缓慢的特点(与其他肌球蛋白II亚型相比)，而且与迄今研究的所有其他肌球蛋白II亚型一样，它们都受到磷酸盐释放的限制。肌球蛋白IIA和IIB的不同寻常之处在于，肌动蛋白对其ADP亲和力没有很大影响，反之亦然。肌球蛋白IIB非常不寻常，因为它从肌动球蛋白释放ADP的速度只比磷酸盐释放速度慢三倍。模拟表明，占空比(在高肌动蛋白亲和力AM.ADP或AM状态下花费的动力学循环的分数)为0.2。这是介于所有其他肌球蛋白II亚型的0.02-0.05和肌球蛋白V或肌球蛋白VI等进行性肌球蛋白的0.6-0.8之间的中间值。这意味着肌球蛋白IIB的功能可能是保持或维持细胞的张力。

Smooth muscle and nonmuscle myosin II are regulated by phosphorylation of the 20 kDa regulatory light chain (RLC) located in the neck region. This region contains a single alpha-helical segment of the myosin heavy chain and the RLC and the essential light chain (ELC). There are three vertebrate genes encoding for nonmuscle myosin IIs, termed IIA and IIB. Platelets express only the IIA isoform, whereas brain expressed predominantly the IIB isoform. Most other cell express nearly equal ratios of IIA and IIB where they are differentially localized. Previous work in our lab has revealed that nonmuscle myosin IIA and myosin IIB have differing enzymatic and motile properties. In order to better understand the basis for this difference, we have engineered myosin IIA and myosin IIB S-1 like fragments for baculviral expression. We have fully characterized the transient enzyme kinetics of these two molecules in the presence and absence of actin. Both are characterized by generally slow kinetics (compared to other myosin II isoforms), and each are rate limited by phosphate release in common with all other myosin II isoforms studied to date. Both myosin IIA and IIB are unusual in that actin does not have a great affect on its ADP affinity and vice versa. Myosin IIB is very unusual in that its ADP release from actomyosin is only three times slower than the rate of phosphate release. Simulations show that the duty ratio (fraction of kinetic cycle spent in high actin affinity AM.ADP or AM states) is 0.2. This is an intermediate value between the usuall 0.02-0.05 found for all other myosin II isoforms and the value of 0.6-0.8 found for processive myosins such as myosin V or myosin VI. This implies that the function of myosin IIB is cells may be to hold or maintain tension.