Fujita K, Ye L H, Sato M, Okagaki T, Nagamachi Y, Kohama K
Department of Pharmacology, Gunma University School of Medicine, Maebashi, Japan.
Mol Cell Biochem. 1999 Jan;190(1-2):85-90.
Myosin light chain kinase (MLCK) has been purified from various muscles as an enzyme to phosphorylate myosin light chains. While the regulatory role of smooth muscle MLCK is well understood, the role of skeletal muscle MLCK in the regulation of contraction has not been fully characterized. Such characterization of skeletal muscle MLCK is difficult because skeletal muscle myosin interacts with actin whether or not the myosin is phosphorylated. Taking the hint from our recent finding that smooth muscle MLCK inhibits the actin-myosin interaction by binding to actin (Kohama et al., Biochem Biophys Res Commun 184: 1204-1211, 1992), we investigated the regulatory role of the actin-binding activity of MLCK from chicken breast muscle in the actin-myosin interaction. The amount of MLCK that bound to actin increased with increases in the concentration of MLCK. However, MLCK hardly bound to myosin. The actin-binding activity of MLCK was affected when Ca2+ and calmodulin (Ca2+ -CaM) were present. The effect of MLCK on the actin-myosin interaction was examined by an in vitro motility assay; the movement of actin-filaments on a myosin-coated glass surface was inhibited by increasing the concentration of MLCK. When CaM was present, the inhibition was overcome in a Ca2+ -dependent manner at microM levels. The inhibition of the movement by MLCK and the recovery from the inhibition by Ca2+ -CaM were not altered whether we use phosphorylated or unphosphorylated myosin for the assay, ruling out the involvement of the kinase activity of MLCK.
肌球蛋白轻链激酶（MLCK）已从各种肌肉中纯化出来，作为一种使肌球蛋白轻链磷酸化的酶。虽然平滑肌MLCK的调节作用已得到充分理解，但骨骼肌MLCK在收缩调节中的作用尚未完全明确。骨骼肌MLCK的这种特性难以确定，因为无论肌球蛋白是否磷酸化，骨骼肌肌球蛋白都能与肌动蛋白相互作用。基于我们最近发现平滑肌MLCK通过与肌动蛋白结合来抑制肌动蛋白 - 肌球蛋白相互作用（小滨等人，《生物化学与生物物理研究通讯》184: 1204 - 1211, 1992）这一发现，我们研究了鸡胸肌MLCK的肌动蛋白结合活性在肌动蛋白 - 肌球蛋白相互作用中的调节作用。与肌动蛋白结合的MLCK量随MLCK浓度的增加而增加。然而，MLCK几乎不与肌球蛋白结合。当存在Ca2 +和钙调蛋白（Ca2 + - CaM）时，MLCK的肌动蛋白结合活性会受到影响。通过体外运动分析检测了MLCK对肌动蛋白 - 肌球蛋白相互作用的影响；在肌球蛋白包被的玻璃表面上，肌动蛋白丝的运动随着MLCK浓度的增加而受到抑制。当存在CaM时，在微摩尔水平上以Ca2 +依赖的方式克服了这种抑制。无论我们在分析中使用磷酸化还是未磷酸化的肌球蛋白，MLCK对运动的抑制以及Ca2 + - CaM对抑制的恢复都没有改变，排除了MLCK激酶活性的参与。
