CHARACTERIZATION OF CALPONIN BINDING TO ACTIN

Calponin, a protein isolated from smooth muscle and nonmuscle cells, h as previously been shown to inhibit the actin-activated ATPase activit y of myosin. Reports of the stoichiometry of binding range from 1 calp onin per actin to 1 calponin per 3 actin monomers. We now report a det ailed study of the binding of [C-14]iodoacetamide-labeled calponin to actin. The labeling procedure did not significantly alter the binding constant of calponin to actin. The stoichiometry of binding was variab le and dependent on ionic strength. Below 110 mM ionic strength, the s toichiometry of binding was 1:1. As the ionic strength was increased a bove 110 mM ionic strength, the stoichiometry shifted from 1:1 to 1 ca lponin per 2 actin monomers. At physiological ionic strength, the bind ing exhibited a small degree of positive cooperativity and was adequat ely described by a single class of binding sites with an association c onstant of 6 x 10(6) M(-1). The affinity decreased to 20% of this valu e in the presence of ATP. Irrespective of the ionic strength, actin fo rmed bundles when saturation with calponin exceeded about 30%. Measure ments of the rate of association were complicated by this bundling, bu t the upper limit for this reaction was placed at 10(6) M(-1) s(-1). T he addition of calponin to actin-caldesmon complexes caused displaceme nt of the caldesmon.