The effect of heavy chain phosphorylation and solution conditions on the assembly of Acanthamoeba myosin-II.
At low ionic strength, Acanthamoeba myosin-II polymerizes into bipolar minifilaments, consisting of eight molecules, that scatter about three times as much light as monomers. With this light scattering assay, we show that the critical concentration for assembly in 50-mM KCl is less than 5 nM. Phosphorylation of the myosin heavy chain over the range of 0.7 to 3.7 P per molecule has no effect on its KCl dependent assembly properties: the structure of the filaments, the extent of assembly, and the critical concentration for assembly are the same. Sucrose at a concentration above a few percent inhibits polymerization. Millimolar concentrations of MgCl2 induce the lateral aggregation of fully formed minifilaments into thick filaments. Compared with dephosphorylated minifilaments, minifilaments of phosphorylated myosin have a lower tendency to aggregate laterally and require higher concentrations of MgCl2 for maximal light scattering. Acidic pH also induces lateral aggregation, whereas basic pH leads to depolymerization of the myosin-II minifilaments. Under polymerizing conditions, millimolar concentrations of ATP only slightly decrease the light scattering of either phosphorylated or dephosphorylated myosin-II. Barring further modulation of assembly by unknown proteins, both phosphorylated and dephosphorylated myosin-II are expected to be in the form of minifilaments under the ionic conditions existing within Acanthamoeba.
Sinard
J H
JH
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Pollard
T D
TD
D000048:Acanthamoeba / Q000378:metabolism*; D000818:Animals; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D015636:Magnesium Chloride / Q000494:pharmacology; D008854:Microscopy, Electron; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*; D009994:Osmolar Concentration; D010766:Phosphorylation; D012542:Scattering, Radiation
D046911:Macromolecular Substances; D015636:Magnesium Chloride; D009218:Myosins