The Effect Of Myosin Light Chain Kinase On Phosphorylated Myosin ATPase Activity And The Velocity Of Actin Filaments In Myosin Molecular By In Vitro Motility

Objective: Myosin light chain kinase (MLCK) is a multifunctional regulatory protein of smooth muscle contraction, which includes the N-terminal actin-binding domain, the central catalytic domain, calmodulin-binding domain, and the C- terminal of myosin-binding domain. The C terminus of MLCK is present in smooth muscle cells as a gene product called telokin, which is independent of MLCK expression. Myosin phosphorylated by the catalytic domain of MLCK is in an active form and interacts with actin filament to contract smooth muscle. This mode of phosphorylation is widely accepted as the regulatory path for actin-myosin interaction. However, there are a number of observations that are not explained by the mode. We have previously engineered an MLCK C-terminal fragment (MLCK860/1176)containing the myosin-binding domain but devoid of a catalytic domain , which have confirmed how myosin is stimulated by this non-kinase pathway. We argued that whether the phosphorylated myosin is also stimulated by this non-kinase pathway.Methods: 1. To culture Bl21 cells and induce protein expression. 2. To prepare and purify protein. 3. To measure the phosphorylated myosin ATPase activity as well as its proteolytic fragment HMM, S1 with EnzChek Phosphate Assay Kit, and implement in vitro motility assay.Results: 1. The Effect of MLCK860/1176 fragment on phosphorylated myosin, HMM and S1 ATPase activity. The results show that the phosphorylated myosin ATPase activity enhances in response to increasing concentration of MLCK860/1176 fragment in the presence of actin with Vmax = 19.426±1.669 (n=3) and Km = 0.486±0.106(n=3);in...