Ye L H, Hayakawa K, Lin Y, Okagaki T, Fujita K, Kohama K
Department of Pharmacology, Gunma University School of Medicine.
J Biochem. 1994 Dec;116(6):1377-82. doi: 10.1093/oxfordjournals.jbchem.a124690.
Myosin light chain kinase (MLCK) is present in muscle cells including those of smooth muscle as an actin-binding protein. By avoiding complication introduced as a result of kinase activity of MLCK, we have demonstrated regulatory role of MLCK through its actin-binding activity [Kohama et al. (1992) Biochem. Biophys. Res. Commun. 184, 1204-1211]. To analyze such a regulatory role of MLCK, we compared the effects of MLCK on the velocity of the movement of actin filaments on a surface coated with smooth muscle myosin with those of another actin-binding proteins in smooth muscle, namely, caldesmon (CaD) and calponin (CaP). Both CaD and CaP stimulated movement when their concentrations were low, but they inhibited movement as their concentrations were increased. Calmodulin (CaM) in the presence of Ca2+ (Ca-CaM) antagonized the inhibition but hardly affected the stimulation. The effect of MLCK, by contrast, was simply inhibitory when Ca-CaM was not present. No stimulation was observed until Ca-CaM was added. The inhibitory ability of these actin-binding proteins increased in the following order: CaD < CaP < MLCK. The effect of MLCK and CaD on movement was further examined on surfaces coated with skeletal muscle myosin. The basic effect was similar to that observed with smooth muscle myosin. However, 10-fold greater concentrations of MLCK and CaD were required for a comparable effect. Such an increase in the required concentration was also observed when the velocity of movement was increased by elevation of the temperature during the assay with smooth muscle myosin. Thus, it is the velocity of movement itself that determines the required concentrations of MLCK and CaD.
肌球蛋白轻链激酶（MLCK）作为一种肌动蛋白结合蛋白存在于包括平滑肌细胞在内的肌肉细胞中。通过避免因MLCK激酶活性而引入的并发症，我们已经证明了MLCK通过其肌动蛋白结合活性发挥的调节作用[小滨等人（1992年），《生物化学与生物物理研究通讯》，184卷，1204 - 1211页]。为了分析MLCK的这种调节作用，我们比较了MLCK对涂有平滑肌肌球蛋白的表面上肌动蛋白丝运动速度的影响与平滑肌中其他肌动蛋白结合蛋白（即钙调蛋白（CaD）和钙泊宁（CaP））的影响。当CaD和CaP的浓度较低时，它们都会刺激运动，但随着浓度增加，它们会抑制运动。在Ca2 +存在的情况下（Ca - CaM），钙调蛋白（CaM）可拮抗这种抑制作用，但对刺激作用几乎没有影响。相比之下，当不存在Ca - CaM时，MLCK的作用只是抑制性的。在添加Ca - CaM之前未观察到刺激作用。这些肌动蛋白结合蛋白的抑制能力按以下顺序增加：CaD < CaP < MLCK。我们进一步在涂有骨骼肌肌球蛋白的表面上研究了MLCK和CaD对运动的影响。基本效果与在平滑肌肌球蛋白上观察到的相似。然而，要达到类似的效果，需要MLCK和CaD的浓度高10倍。当在平滑肌肌球蛋白测定过程中通过升高温度来提高运动速度时，也观察到了所需浓度的这种增加。因此，是运动速度本身决定了MLCK和CaD的所需浓度。
