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J Biol Chem. 2014 Aug 22;289(34):23977-91. doi: 10.1074/jbc.M114.562231. Epub 2014 Jul 8.

Magnesium modulates actin binding and ADP release in myosin motors.

Author information

1
From the Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033.
2
the Department of Biochemistry, Eötvös Loránd University, H-1117 Budapest, Hungary.
3
the Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405.
4
the Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
5
the Department of Biochemistry, Eötvös Loránd University, H-1117 Budapest, Hungary, the Hungarian Academy of Sciences-Eötvös Loránd University Molecular Biophysics Research Group, H-1117 Budapest, Hungary.
6
the Department of Kinesiology, University of Massachusetts, Amherst, Massachusetts 02210, and.
7
From the Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, cyengo@hmc.psu.edu.

Abstract

We examined the magnesium dependence of five class II myosins, including fast skeletal muscle myosin, smooth muscle myosin, β-cardiac myosin (CMIIB), Dictyostelium myosin II (DdMII), and nonmuscle myosin IIA, as well as myosin V. We found that the myosins examined are inhibited in a Mg(2+)-dependent manner (0.3-9.0 mm free Mg(2+)) in both ATPase and motility assays, under conditions in which the ionic strength was held constant. We found that the ADP release rate constant is reduced by Mg(2+) in myosin V, smooth muscle myosin, nonmuscle myosin IIA, CMIIB, and DdMII, although the ADP affinity is fairly insensitive to Mg(2+) in fast skeletal muscle myosin, CMIIB, and DdMII. Single tryptophan probes in the switch I (Trp-239) and switch II (Trp-501) region of DdMII demonstrate these conserved regions of the active site are sensitive to Mg(2+) coordination. Cardiac muscle fiber mechanic studies demonstrate cross-bridge attachment time is increased at higher Mg(2+) concentrations, demonstrating that the ADP release rate constant is slowed by Mg(2+) in the context of an activated muscle fiber. Direct measurements of phosphate release in myosin V demonstrate that Mg(2+) reduces actin affinity in the M·ADP·Pi state, although it does not change the rate of phosphate release. Therefore, the Mg(2+) inhibition of the actin-activated ATPase activity observed in class II myosins is likely the result of Mg(2+)-dependent alterations in actin binding. Overall, our results suggest that Mg(2+) reduces the ADP release rate constant and rate of attachment to actin in both high and low duty ratio myosins.

KEYWORDS:

Actin; Contractile Protein; Enzyme Kinetics; Magnesium; Muscle; Myosin

PMID:
25006251
PMCID:
PMC4156094
DOI:
10.1074/jbc.M114.562231
[Indexed for MEDLINE]
Free PMC Article
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