Purification and characterization of actin-activatable, Ca2+-sensitive myosin II from Acanthamoeba.
Approximately 8-10 mg of highly actin-activatable, CA2+-sensitive Acanthamoeba myosin II can be isolated in greater than 98% purity from 100 g of amoeba by the new procedure described in detail in this paper. The enzyme isolated by this procedure can be activated by actin because its heavy chains are not fully phosphorylated (Collins, J. H., and Korn, E. D. (1980) J. Biol Chem. 255, 8011-8014). We now show that Acanthamoeba myosin II Mg2+-ATPase activity is more highly activated by Acanthamoeba actin than by muscle actin. Also, actomyosin II ATPase is inactive at concentrations of free Mg2+ lower than about 3 mM and fully active at Mg2+ concentrations greater than 4 mM. Actomyosin II Mg2+-ATPase activity is stimulated by micromolar Ca2+ when assayed over the narrow range of about 3-4 mM Mg2+ but is not affected by Ca2+ at either lower or higher concentrations of Mg2+. The specific activity of te actomyosin II Mg2+-ATPase also increases with increasing concentrations of myosin II when the free Mg2+ concentration is in the range of 3-4 mM but is independent of the myosin II concentration at lower or higher concentrations of Mg2+ . This marked effect of the Mg2+ concentration on the Ca2+-sensitivity and myosin concentration-dependence of th specific activity of actomyosin II ATPase activity does not seem to be related to the formation of myosin filaments, and to be related to the formation of myosin filaments, and myosin II is insoluble only at high concentrations of free Mg2+ (6-7 mM) were neither of these effects is observed. Also, the Mg2+ requirements for actomyosin II ATPase activity and myosin II insolubility can be differentially modified by EDTA and sucrose.
D000199:Actins / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000656:Amoeba / Q000201:enzymology*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D009218:Myosins / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits
D000199:Actins; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins; D000252:Calcium-Transporting ATPases; D008274:Magnesium