Cross R A, Sobieszek A
FEBS Lett. 1985 Sep 2;188(2):367-74. doi: 10.1016/0014-5793(85)80404-x.
Conventional smooth muscle myosin preparations contain a tightly bound myosin light chain kinase activity, which is incompletely removed by gel filtration at high ionic strength. We show here that by contrast, this kinase activity is released, together with calmodulin, under conditions in which myosin is in the folded configuration. The conformation-related release of kinase occurred for dephosphorylated myosin in both the presence and absence of ATP and Ca2+. Binding of kinase to extended phosphorylated myosin was relatively weaker than to dephosphorylated myosin, but was nonetheless detected. The kinetic consequences of this binding behaviour were determined by measuring initial myosin phosphorylation rates as a function of KCl concentration. Rate optima occurred at 60 mM KCl and 300 mM KCl, conditions favouring respectively stable filaments and stable extended monomers. Phosphorylation of the folded monomer was uniformly slow at low KCl concentrations. The folded myosin monomer is thus a relatively poor substrate for the kinase, and is therefore unlikely to represent an analog of the relaxed crossbridge configuration in myosin filaments.
传统的平滑肌肌球蛋白制剂含有紧密结合的肌球蛋白轻链激酶活性，在高离子强度下通过凝胶过滤不能完全去除该活性。我们在此表明，相比之下，在肌球蛋白处于折叠构象的条件下，这种激酶活性会与钙调蛋白一起释放。激酶与构象相关的释放发生在有和没有ATP及Ca2+的情况下的去磷酸化肌球蛋白中。激酶与伸展的磷酸化肌球蛋白的结合相对弱于与去磷酸化肌球蛋白的结合，但仍可检测到。通过测量初始肌球蛋白磷酸化速率作为KCl浓度的函数来确定这种结合行为的动力学后果。最佳速率出现在60 mM KCl和300 mM KCl时，这两种条件分别有利于稳定的细丝和稳定的伸展单体。在低KCl浓度下，折叠单体的磷酸化一直较慢。因此，折叠的肌球蛋白单体是激酶相对较差的底物，因此不太可能代表肌球蛋白细丝中松弛横桥构象的类似物。
