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J. Biol. Chem., Vol. 265, Issue 25, 14864-14869, Sep, 1990
S Umemoto and JR Sellers
We have used two in vitro motility assays to study the relative movement of
actin and myosin from turkey gizzards (smooth muscle) and human platelets.
In the Nitella-based in vitro motility assay, myosin- coated polymer beads
move over a fixed substratum of actin bundles derived from dissection of
the alga, Nitella, whereas in the sliding actin filament assay
fluorescently labeled actin filaments slide over myosin molecules adhered
to a glass surface. Both assay systems yielded similar relative velocities
using smooth muscle myosin and actin under our standard conditions. We have
studied the effects of ATP, ionic strength, magnesium, and tropomyosin on
the velocity and found that with the exception of the dependence on MgCl2,
the two assays gave very similar results. Calcium over a concentration of
pCa 8 to 4 had no effect on the velocity of actin filaments. Phosphorylated
smooth muscle myosin propelled filaments of smooth muscle and skeletal
muscle actin at the same rate. Phosphorylated smooth muscle and cytoplasmic
myosin monomers also moved actin filaments, demonstrating that filament
formation is not required for movement.
Characterization of in vitro motility assays using smooth muscle and cytoplasmic myosins
Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
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