摘要
肌球蛋白轻链激酶(myosin light chain kinase,MLCK)具有激酶和非激酶活性,在平滑肌收缩过程中起着关键酶调控的作用.为进一步阐明MLCK非激酶活性在平滑肌收缩过程中的调节作用,利用已删除部分激酶区域的MLCK重组体(pGEX-F6.5)在大肠杆菌中进行表达,采用亲和层析技术纯化表达的MLCK片段,应用EnzChek磷分析试剂盒检测MLCK片段对磷酸化肌球蛋白、水解重酶解肌球蛋白(heavy meromyosin,HMM)及肌球蛋白亚片段1(subfragment1,S1)ATP酶活性的影响,体外检测MLCK片段对肌动蛋白肌丝运动的调节.研究结果显示,pGEX-F6.5重组表达载体在大肠杆菌中以可溶性GST融合蛋白的形式表达.该融合蛋白经Glutathione-Sepharose4B纯化、SDS-PAGE鉴定得到较纯的单一表达条带.纯化的MLCK片段对磷酸化肌球蛋白、HMM和S1的ATP酶活性均有明显激活作用.MLCK片段激活磷酸化肌球蛋白ATP酶活性为:Vmax=(19.426±1.669)倍;Km=(0.486±0.106)μmol/L,MLCK片段对磷酸化HMM和S1的ATP酶活性也有相似的刺激作用.体外肌丝运动研究表明,随着MLCK片段浓度的增加,磷酸化肌球蛋白与肌动蛋白结合的数量不断增加,肌丝运动的速度也随之增加.上述结果表明,MLCK的C端非激酶活性具有调节磷酸化的肌球蛋白ATP酶活性及肌丝运动的作用.
Myosin light chain kinase （MLCK） is a multif unctional regulatory protein of smooth muscle contraction, which includes an N-terminal actin-binding domain, central catalytic domain, calmodulin-binding domain, and a C- terminal myosin-binding domain. Myosin phosphorylated by the catalytic domain of MLCK is in an active form and interacts with the actin filament to contract smooth muscle. This mode of phosphorylation is widely accepted as the regulatory mechanism for actin-myosin interaction. However, there are a number of observations that are not explained by this mechanism. An MLCK C-terminal fragment （MLCK fragment） containing the myosin-binding domain have been previously engineered but devoid of a catalytic domain, which has confirmed how myosin is stimulated by this non-kinase pathway. A recombinant GST-fusion protein of the MLCK fragment was expressed in E.coli and detected by SDS-PAGE. Through Glutathione-Sepharose 4B affinity chromatography, a single pure band of the MLCK fragment was obtained. The phosphorylated myosin ATPase activities of the MLCK fragment, as well as its proteolytic fragment HMM and S l were measured with the EnzChek Phosphate Assay Kit. The MLCK fragment stimulated phosphorylated myosin ATPase activity （Vmax= （19.426 ±1.669）-fold, Km=（0.486 ±0.106）μmol/L）. Similar stimulation figures were obtained by measuring the ATPase activity of phosphorylated HMM and S I. The data suggests that MLCK could activate phosphorylated myosin, HMM and Sl. In addition, in vitro motility assays demonstrated that increasing amounts of the MLCK fragment increased actin-myosin interaction and sliding. Also, the velocity of actin filaments could be enhanced on a gizzard smooth muscle myosin surface with the MLCK fragment. It is conclude that the non-kinase C-terminal domain of MLCK is independent of the phosphorylating mode for activation of myosin.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2008年第1期91-96,共6页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金资助项目(30470394).~~
