Characterization of calponin binding to actin.
Calponin, a protein isolated from smooth muscle and nonmuscle cells, has previously been shown to inhibit the actin-activated ATPase activity of myosin. Reports of the stoichiometry of binding range from 1 calponin per actin to 1 calponin per 3 actin monomers. We now report a detailed study of the binding of [14C]iodoacetamide-labeled calponin to actin. The labeling procedure did not significantly alter the binding constant of calponin to actin. The stoichiometry of binding was variable and dependent on ionic strength. Below 110 mM ionic strength, the stoichiometry of binding was 1:1. As the ionic strength was increased above 110 mM ionic strength, the stoichiometry shifted from 1:1 to 1 calponin per 2 actin monomers. At physiological ionic strength, the binding exhibited a small degree of positive cooperativity and was adequately described by a single class of binding sites with an association constant of 6 x 10(6) M-1. The affinity decreased to 20% of this value in the presence of ATP. Irrespective of the ionic strength, actin formed bundles when saturation with calponin exceeded about 30%. Measurements of the rate of association were complicated by this bundling, but the upper limit for this reaction was placed at 10(6) M-1 s-1. The addition of calponin to actin-caldesmon complexes caused displacement of the caldesmon.
Lu
F W
FW
Department of Biochemistry, East Carolina University School of Medicine, Greenville, North Carolina 27858-4354, USA.
Freedman
M V
MV
Chalovich
J M
JM
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D007460:Iodoacetamide / Q000378:metabolism; D007700:Kinetics; D008840:Microfilament Proteins; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D009994:Osmolar Concentration; D011485:Protein Binding; D011817:Rabbits; D000096985:Calponins
D000199:Actins; D000255:Adenosine Triphosphate; D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; D007460:Iodoacetamide; D008840:Microfilament Proteins; D009218:Myosins; D000096985:Calponins