Modulatory Effect of MgATP on Smooth Muscle Myosin Phosphatase Activity
To deepen our understanding of the regulatory mechanism of smooth muscle contraction, we examined properties of smooth muscle myosin phosphatase (SMMP) which was purified from chicken gizzard according to the method of Alessi et al. with slight modifications. The SMMP was a heterotrimer of 130, 37 and 20 kDa subunits as reported. Because the enzymatic activity was strongly dependent on the ionic strength, all experiments were carried out at a constant ionic strength of 0.15 M. The Vmax and Km of the enzyme toward bovine stomach phosphorylated myosin was 25.2 μmol/mg protein/min and 0.45 μM at 25°C, respectively, which implied that the rate of dephosphorylation by SMMP in smooth muscle cells should be comparable to that of phosphorylation by the myosin light chain kinase in the presence of a saturating Ca2+ concentration. ATP dose-de-pendently decreased the SMMP activity to one-fifth. The IC50 for ATP was 0.6 uM in the presence of 5 mM MgCl2, whereas it increased to 10 μM in the absence of Mg2+ with the addition of 1 mM EDTA. ADP, AMP, GTP and ITP showed no or only weak effect. The finding that ATPγS was ineffective indicates that phosphorylation of the 130 kDa subunit cannot be the underlying mechanism. This modulation was observed with myosin from bovine stomach, but not from chicken gizzard. The difference in conformation of myosin molecules in the presence of MgATP may be critical in its modulatory effect.
KeywordsMyosin Light Chain Myosin Light Chain Kinase Constant Ionic Strength Myosin Molecule Myosin Phosphatase
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