title: Interdependence of factors affecting the actin-activated ATPase activity of myosin II from Acanthamoeba castellanii.
abstract: Acanthamoeba myosin II can be phosphorylated at three serine residues near the C terminus of each heavy chain. Deophosphorylated myosin II has the highest actin-activated ATPase activity. In this paper, we report the interdependent effects of phosphorylation, Mg2+, Ca2+, temperature, pH, and KCl on the acti-activated ATPase activity. With increasing level of phosphorylation, the actin-activated ATPase activity decreases and the optimal concentration of Mg2+ increases. Lowering the temperature of assay from 35 to 20 degrees C reduces the specific activity of dephosphorylated myosin II and increases the optimal Mg2+ concentration. Lowering the pH from 7.7 to 6.4 decreases the optimal Mg2+ concentration for dephosphorylated myosin II but has no effect on its specific activity. Below pH 6.4, the activity of dephosphorylated myosin is decreased. Phosphorylated myosin II, on the other hand, has no actin-activated ATPase activity at pH 6.4 and above, irrespective of the Mg2+ concentration, but has significant activity at lower pH with a maximum at pH 6.0-6.1 in 1 mM Mg2+. Dephosphorylated myosin II requires Mg2+ for actin-activated ATPase activity under all conditions, but Ca2+ can substitute for some of the Mg2+ at pH 7.0. Partial inhibition of dephosphorylated myosin II by 10-15 mM KCl can be overcome by increasing the Mg2+ concentration but the enzyme is 60% inhibited at 25 mM KCl irrespective of the Mg2+ concentration. The actin-activated ATPase activity of maximally dephosphorylated myosin II is as high at pH 6.4, 1 mM Mg2+, and 30 degrees C, which may be near physiological conditions, as at pH 7, 4 mM Mg2+, and 35 degrees C, the assay conditions commonly used previously. Under both conditions, maximally phosphorylated myosin II is inactive. The interdependence of all these effectors emphasizes the ned to employ multiple incubation conditions in assessing the actin-activated ATPase activities of myosins from all sources.
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000656:Amoeba / Q000201:enzymology*; D000818:Animals; D002413:Cations, Divalent; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium