Characterization of cytoplasmic actin isolated from Acanthamoeba castellanii by a new method.
Cytoplasmic actin has been isolated from Acanthamoeba castellanii by a new method, employing chromatography on DEAE-cellulose, that improves the yield by more than 20-fold over the previously reported method. This procedure should be particularly useful for isolating actin from cells in which it is present in relatively low concentration because the method does not depend initially on the polymerization of actin or its interaction with myosin. Systematic comparison of the properties of purified Acanthamoeba actin and rabbit skeletal muscle actin shows them to be similar in many ways: viscosity of F-actin, stoichiometry of bound nucleotide, stoichiometry of binding of muscle heavy meromyosin and myosin subfragment 1 in the absence of ATP, and ability to inhibit the KATPase activity of heavy meromyosin. The amino acid compositions of Acanthamoeba and muscle actin are also quite similar, but significant differences, especially the presence of epsilon-N-methyllysines in Acanthamoeba actin, have been confirmed. In addition to this structural difference, we find that Acanthamoeba actin is only one-third as effective as muscle actin as an activator of the MgATPase of muscle heavy meromyosin and subfragment 1. For Acanthamoeba actin, as for muscle actin, this activation exhibits hyperbolic dependence on actin concentration; i.e. the double reciprocal plot of ATPase activation versus actin concentration is linear. From these plots we find that the two actins give the same extrapolated ATPase activity at infinite actin concentration (Vmax) but differ by a factor of three in the concentration of actin needed to produce half-maximal activation (Kapp).
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000656:Amoeba / Q000378:metabolism*; D000818:Animals; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000032:analysis; D011817:Rabbits; D014783:Viscosity
D000199:Actins; D000596:Amino Acids; D000251:Adenosine Triphosphatases; D008274:Magnesium