平滑肌肌球蛋白轻链激酶及ATP结合位点突变体对ATP酶活性的调节作用  

Effect of Smooth Muscle Myosin Light Chain Kinase and the ATP Binding Site Mutant on ATPase Activity

作　　者：崔颖[1] 谢策[2] 吕广艳[1] 王辉[1] 赵莹[1] 魏晓晴[1] 曲淑贤[1] 曹晶萍[1] 高颖[1,2] 

机构地区：[1]辽宁省医学细胞分子生物学重点实验室,辽宁大连116044 [2]大连医科大学生化教研室,辽宁大连116044

出　　处：《现代生物医学进展》2009年第16期3034-3037,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金资助项目(30470394)

摘　　要：目的:探寻MLCK的非激酶活性区域对MLCK活性的影响,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。方法:利用编码MLCK全长的pColdI表达载体对其ATP结合位点进行定点突变,获得无激酶活性的MLCK突变体;应用Glycerol-PAGE鉴定肌球蛋白磷酸化水平;应用孔雀绿方法检测重组MLCK对肌球蛋白ATP酶活性的影响。结果:MLCK/△ATP(突变型)失去磷酸化肌球蛋白轻链的激酶活性;重组MLCK(野生型)和MLCK/△ATP(突变型)均可以在非钙条件下激活非磷酸化肌球蛋白Mg2+-ATP酶活性,抑制磷酸化肌球蛋白的Mg2+-ATP酶活性,而且激活与抑制作用均随着MLCK浓度的增加而增大,但二者对肌球蛋白的ATP酶活性的作用没有显著差异(P>0.05)。结论:平滑肌肌球蛋白轻链激酶及ATP结合位点突变体具有激活非磷酸化肌球蛋白ATP酶活性的作用。Objective： To investigate the non-kinase activity of MLCK and to explain the molecular mechanismof its regulating in Smooth muscle contraction. Methods： The pCold I expression vector containing the cDNA of full length MLCK was used to get mutation and MLCK without the kinase activity were acquired. Glycerol-PAGE was used to confirm the phosphorylated myosin light chain. Marachite green method was used to examine the effect of recombinant full-length MLCKs on the phosphorylated and unphosphorylated myosin. Results： The MLCK/△ATP lost its kinase activity of phosphorylating myosin light chain. Both the wild type of recombinant MLCK and the MLCK/△ATP could activate the unphosphorylated myosin Mg2＋-ATPase activity and inhibit the phosphorylated myosin Mg2＋-ATPase activity in a dose dependent manner when there was no Ca2＋ existing. But there was no prominent difference between the wild type of recombinant MLCK and the MLCK/△ATP on the myosin ATPase activity （P〉0.05）. conclusions Both full-length smooth muscle myosin light chain kinase and its ATP binding site mutant could active unphosphorylated myosin ATPase activity.

关 键 词：平滑肌收缩 肌球蛋白轻链激酶(MLCK) 肌球蛋白 肌球蛋白Mg2+-ATP酶活性 

分 类 号：Q71[生物学—分子生物学]