肌球蛋白轻链激酶活性片段对肌球蛋白轻链的非钙依赖性磷酸化作用特征

肌球蛋白轻链激酶 (MLCK)的活性片段 (MLCKF)能比完整的MLCK更有效地、以非钙依赖性的方式磷酸化肌球蛋白轻链 (MLC2 0 )。该片段是用胰蛋白酶水解MLCK ,再经DEAE 5 2柱层析分离而获得的 ,分子量约为 6 1kD。Western印迹已证实该MLCKF与完整的MLCK同源。MLCKF对肌球蛋白轻链的磷酸化作用及其作用特征通过甘油电泳及ScoinImage扫描软件检测 ,肌球蛋白ATP酶活性通过分光光度法检测。实验结果证实 ,MLCKF催化的MLC2 0 非钙依赖性磷酸化 (CIPM)比MLCK催化的CIPM效力高、耗能多 ,但比MLCK催化的MLC2 0 钙依赖性磷酸化 (CDPM)效力低、耗能少 ;MLCKF催化的CIPM与MLCK催化的CIPM均较MLCK催化的CDPM稳定 ,不易受温育温度、温育时间及离子浓度等变化的影响 ,且对MLCK抑制剂ML 9敏感性低。

The Characterization of Ca ~(2 )-calmodulin Independent Phosphorylation of Myosin Light Chains by a Fragment from Myosin Light Chain Kinase

A constitutively active myosin light chain kinase (MLCK) fragment (MLCKF) was found to phosphorylate myosin light chains (MLC 20) in a Ca 2 -CaM independent way more effectively than the intact MLCK. The MLCKF was prepared by tryptic digestion of MLCK. Western blot was used to demonstrate the homogeneity of trypsin-digested MLCKF and intact MLCK. Phosphorylation of MLC 20 was detected by Gly-PAGE and Scoin Image Software, and Mg 2 -ATPase activity of myosin was measured with spectrophotometry. Our results indicated that Ca 2 -CaM independent phosphorylation of myosin (CIPM) by MLCKF was more efficient than CIPM by MLCK and less efficient than Ca 2 -CaM dependent phosphorylation of myosin (CDPM) by MLCK in phosphorylating MLC 20 and stimulating myosin Mg 2 -ATPase activity; both CIPM by MLCKF and CIPM by MLCK were less influenced by the rise of incubation-temperature, the prolonging of incubation-time, the increase of ionic strength of KCl and less sensitive to MLCK inhibitor ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine] than CDPM by MLCK. The differences were statistically significant (P<0.01, or P<0.05). The results may be valuable to further investigating the mechanisms of sustained tension characterized by less energy consumption.