平滑肌肌球蛋白轻链激酶的非激酶作用及对ATP 酶活性的调节

肌球蛋白轻链激酶（myosin light chain kinase, MLCK）具有激酶活性和非激酶活性，在平滑肌收缩过程中起着关键酶调控的作用.为探寻MLCK的非激酶活性区域对MLCK活性的影响，以进一步阐明MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制.采用PCR技术构建MLCK部分氨基酸缺失的重组表达载体pGEX-F6-5/D，经大肠杆菌表达得到可溶性GST融合蛋白，利用SDS-PAGE及Western 印迹鉴定表达的MLCK在细胞中的分布，结果还显示,提取液的上清和沉淀中均有MLCK片段的表达.运用亲和层析技术分离并纯化删除前、后表达的MLCK片段（F6.5和F6-5/D），经谷胱甘肽琼脂糖凝胶 4B 纯化，SDS-PAGE鉴定显示为单一表达条带.应用EnzChek磷分析试剂盒和孔雀绿两种方法分别测定不同浓度的MLCK对非磷酸化肌球蛋白Mg²⁺-ATP酶活性的影响.两种MLCK的片段均具有激活ATP酶活性的作用，并随MLCK浓度的增加，酶的活性增加.比较删除前后不同MLCK片段对ATP酶活性的影响结果显示，删除MLCK片段1002位丙氨酸至1019位亮氨酸后,对ATP酶的激活作用较删除前明显降低，表明删除的部分氨基酸序列为MLCK非激酶活性所必需的区域.利用电镜技术观察到MLCK片段（F6.5）使非磷酸化肌球蛋白构象发生明显的变化.加入MLCK片段后肌球蛋白的构象由非活性型转化为活性型，并且MLCK片段还具有促进肌球蛋白单体形成肌丝的作用.

Effect of Non-kinase Activity of Myosin Light Chain Kinase on ATPase Activity of Smooth Muscle Myosin

Myosin light chain kinase (MLCK) possesses kinase and non-kinase properties, and both properties play important roles in the regulation of smooth muscle contraction. The purpose is to explore the non-kinase activity of MLCK and to elucidate the molecular mechanism of its activity in regulating contraction. A recombinant GST-fusion protein of the C-terminal domain of MLCK (F6.5) was created. The C-terminal fragment of MLCK was truncated further by deletion of the Ca2 /calmodulin domain (F6.5/D) within this fragment. The MLCK constructs were expressed in E.coli and purified. Using the EnzChek Phosphate Assay and the malachite green method, the unphosphorylated myosin ATPase activity of these recombinant proteins was measured. The results showed that both constructs could stimulate ATPase activity in a dose-dependent manner. However, the extent of ATPase activity was much lower in the F6.5/D construct, suggesting that the amino acid sequence Ala1002-Leu1019 was essential for the non-kinase activity of MLCK. Electron microscopy showed that MLCK(F6.5) could transform inactive myosin filaments to the active form and promote myosin filaments bundle formation.