Calponin, a major actin-, tropomyosin-, and calmodulin-binding protein
in smooth muscle, interacted with tubulin, a main constituent of micr
otubules, in a concentration-dependent fashion in, vitro, The apparent
K-d value of calponin to tubulin was calculated to be 5.2 mu M with 2
mol of calponin maximally bound per 1 mol of tubulin, At low ionic st
rength, tubulin bound to calponin immobilized on Sepharose 4B, and the
bound protein was released at about 270 mM NaCl. Chemical cross-linki
ng experiments showed that a 1:1 molar covalent complex of calponin an
d tubulin was produced, The amount of calponin bound to microtubules d
ecreased with increasing ionic strength or Ca2+ concentration. The add
ition of calmodulin or S100 to the mixture of calponin and microtubule
proteins caused the removal of calponin from microtubules in the pres
ence of Ca2+, but not in the presence of EGTA, Calponin-related protei
ns including tropomyosin, SM22, and caldesmon had little effect on the
calponin binding to microtubules, whereas MAP2 inhibited the binding,
Interestingly, there was little, if any, effect of mycalolide B-treat
ed actin on the binding of calponin to microtubules, Furthermore, only
about 20% of calponin-F-actin interaction was inhibited in the presen
ce of an excess amount of tubulin (4 mol per mol of calponin), indicat
ing that tubulin binds to calponin at a different site from that of ac
tin, Compared with MAP2, calponin had little effect on microtubule pol
ymerization.