Objective.To study the exact effects and characteristics of a constitutively active myosin light chain kinase fragment (MLCKF)on Ca 2+ -CaM dependent phosphorylation of myosin light chains(CDPM)of smooth muscle.Method...Objective.To study the exact effects and characteristics of a constitutively active myosin light chain kinase fragment (MLCKF)on Ca 2+ -CaM dependent phosphorylation of myosin light chains(CDPM)of smooth muscle.Methods.Proteolysis of myosin light chain kinase(MLCK)by trypsin was used to obtain the con-stitutively active fragment of MLCK;Western blot was applied to demonstrate the homogeneity of typsin-digested MLCK fragments and intact MLCK.The phosphorylation of myosin light chains was detected us-ing SDS-PAGE and Scoin Image Software,and myosin Mg 2+ -ATPase activities were measured using spec-trophotometry to observe the characteristics of MLCKF in phosphorylating myosin light chains and stimu-lating myosin Mg 2+ -ATPase activities compared with intact MLCK.Results.Our assay demonstrated that MLCKF had a specific activity on CDPM.We also found that the extent of CDPM by MLCKF was greater than that by intact MLCK at different concentrations,differ-ent incubation time and different incubation temperatures.CDPM by MLCKF was also less influenced by the change of ionic strength of KCl than CDPM by intact MLCK.Mg 2+ -ATPase activities of phosphorylat-ed myosin light chains by MLCKF were higher than those by MLCK at different concentrations and dif-ferent reaction time.These differences were statistically significant ( P<0.01, P<0.05).Conclusion.MLCKF not only possessed a specific activity on CDPM but also was more efficient than MLCK in phosphorylating myosin light chains and stimulating myosin Mg 2+ -ATPase activities.Our results suggested that MLCKF possibly plays a certain role in smooth muscle contraction-relaxation cycle.展开更多
文摘Objective.To study the exact effects and characteristics of a constitutively active myosin light chain kinase fragment (MLCKF)on Ca 2+ -CaM dependent phosphorylation of myosin light chains(CDPM)of smooth muscle.Methods.Proteolysis of myosin light chain kinase(MLCK)by trypsin was used to obtain the con-stitutively active fragment of MLCK;Western blot was applied to demonstrate the homogeneity of typsin-digested MLCK fragments and intact MLCK.The phosphorylation of myosin light chains was detected us-ing SDS-PAGE and Scoin Image Software,and myosin Mg 2+ -ATPase activities were measured using spec-trophotometry to observe the characteristics of MLCKF in phosphorylating myosin light chains and stimu-lating myosin Mg 2+ -ATPase activities compared with intact MLCK.Results.Our assay demonstrated that MLCKF had a specific activity on CDPM.We also found that the extent of CDPM by MLCKF was greater than that by intact MLCK at different concentrations,differ-ent incubation time and different incubation temperatures.CDPM by MLCKF was also less influenced by the change of ionic strength of KCl than CDPM by intact MLCK.Mg 2+ -ATPase activities of phosphorylat-ed myosin light chains by MLCKF were higher than those by MLCK at different concentrations and dif-ferent reaction time.These differences were statistically significant ( P<0.01, P<0.05).Conclusion.MLCKF not only possessed a specific activity on CDPM but also was more efficient than MLCK in phosphorylating myosin light chains and stimulating myosin Mg 2+ -ATPase activities.Our results suggested that MLCKF possibly plays a certain role in smooth muscle contraction-relaxation cycle.
