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Modulatory effect of MgATP on smooth muscle myosin phosphatase activity
1Juntendo University School of Medicine, Department of Pharmacology, Tokyo. ysogawa@med.juntendo.ac.jp
Abstract:
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 K(m) of the enzyme toward bovine stomach phosphorylated myosin was 25.2 mumol/mg protein/min and 0.45 microM at 25 degrees 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-dependently decreased the SMMP activity to one-fifth. The IC50 for ATP was 0.6 microM in the presence of 5 mM MgCl2, whereas it increased to 10 microM 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 ATP gamma S 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.
Insights
Smooth muscle myosin phosphatase (SMMP) regulates muscle contraction by dephosphorylating myosin. ATP inhibits SMMP activity, suggesting a key regulatory role in smooth muscle function.
Area of Science:
- Biochemistry
- Molecular Biology
- Physiology
Background:
- Smooth muscle contraction is regulated by myosin phosphorylation.
- Smooth muscle myosin phosphatase (SMMP) plays a crucial role in dephosphorylating myosin.
- Understanding SMMP regulation is key to understanding smooth muscle function.
Purpose of the Study:
- To investigate the properties and regulatory mechanisms of smooth muscle myosin phosphatase (SMMP).
- To determine the kinetic parameters and substrate specificity of SMMP.
- To elucidate the role of ATP and other nucleotides in modulating SMMP activity.
Main Methods:
- Purification of SMMP from chicken gizzard using established methods.
- Enzymatic assays to determine Vmax and Km with phosphorylated bovine stomach myosin.
- Investigating the effects of ATP, ADP, AMP, GTP, and ITP on SMMP activity at constant ionic strength.
Main Results:
- SMMP is a heterotrimer composed of 130, 37, and 20 kDa subunits.
- SMMP exhibited high affinity and catalytic efficiency towards phosphorylated myosin.
- ATP significantly inhibited SMMP activity in a dose-dependent manner, with Mg2+ influencing the IC50.
- ATP gamma S was ineffective, suggesting phosphorylation of the 130 kDa subunit is not the inhibitory mechanism.
Conclusions:
- SMMP activity is modulated by ATP, indicating a regulatory role in smooth muscle contraction.
- The observed inhibition by ATP suggests a mechanism for controlling the balance between phosphorylation and dephosphorylation of myosin.
- Differences in myosin conformation in the presence of MgATP may be critical for SMMP modulation.