Cloning, Expression And Function Analysis Of The Deletion Of Myosin Light Chain Kinase CDNA

The well-established mode for the regulation of smooth muscle contraction is: Ca²⁺ form a complex with calmodulin(CaM), the complex activates myosin light chain kinase (MLCK) to phosphorylate 20kDa myosin light chain (MLC20) at Ser19 to activate myosin ATPase activity,and the inorganic phosphate resulting from Mg²⁺-ATP hydrolyzed by activatied myosin Mg²⁺-ATPase binds with cross-bridge to increase its Gibbs free energy, which initiates cyclic cross-bridge-actin interactions and sliding between thick filaments and thin filaments followed by smooth muscle contraction. However, smooth muscle remains tension at resting cytosolic free Ca²⁺ concentration and its contraction can be detected in the absence of Ca²⁺/CaM, the reason for this phenomenon is unknown well. Myosin light chain kinase of smooth muscle (sm MLCK)plays an important role in regulating smooth muscle contraction as a key enzyme with kinase activity as well as non-kinase activity.To elucidate the molecular mebhanism of MLCK regulating smooth muscle contraction through its non-kinase activity, in this research we constructed a recombinant vector pGEX-F6.5/D for expressing MLCK short of a number of amino acids employing PCR technique through the template pGEX-F6.5, and then expressed in E.coli BL21. The expressed MLCK segments distribution in cell were detected by SDS-PAGE and Western blotting and purified through affinity chromatography. Unphosphorylated myosin Mg²⁺-ATPase activity was measured with...