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.