title: Magnesium modulates actin binding and ADP release in myosin motors.
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.
authors.affiliation: From the Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033.
authors.forename: Anja M
authors.initials: AM
authors.lastname: Swenson
authors.affiliation: From the Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033.
authors.forename: Darshan V
authors.initials: DV
authors.lastname: Trivedi
authors.affiliation: the Department of Biochemistry, Eötvös Loránd University, H-1117 Budapest, Hungary.
authors.forename: Anna A
authors.initials: AA
authors.lastname: Rauscher
authors.affiliation: the Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: the Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Takagi
authors.affiliation: the Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405.
authors.forename: Bradley M
authors.initials: BM
authors.lastname: Palmer
authors.affiliation: 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.
authors.forename: András
authors.initials: A
authors.lastname: Málnási-Csizmadia
authors.affiliation: the Department of Kinesiology, University of Massachusetts, Amherst, Massachusetts 02210, and.
authors.forename: Edward P
authors.initials: EP
authors.lastname: Debold
authors.affiliation: From the Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, cyengo@hmc.psu.edu.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Yengo
mesh_terms: D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D007700:Kinetics; D008274:Magnesium / Q000502:physiology*; D020409:Molecular Motor Proteins / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D013552:Swine
chemical_list: D000199:Actins; D020409:Molecular Motor Proteins; D000244:Adenosine Diphosphate; D009218:Myosins; D008274:Magnesium
keywords: Actin; Contractile Protein; Enzyme Kinetics; Magnesium; Muscle; Myosin