EFFECT OF CALTROPIN ON CALDESMON-ACTIN INTERACTION

The binding of chicken gizzard caldesmon to actin was studied both in the presence and the absence of caltropin using Airfuge centrifugation experiments, disulfide cross-linking studies, and the fluorescent pro be acrylodan (6-acryloyl-2-(dimethylamino)napththalene). In co-sedimen tation studies most of the caldesmon pelleted along with actin. Howeve r, when caldesmon in the presence of caltropin was mixed with actin, c aldesmon did not pellet along with actin following high speed centrifu gation, suggesting that caltropin has significantly weakened its bindi ng to actin. The caltropin effect was noticed even when tropomyosin wa s included in the reaction mixture. Acrylodan-labeled caldesmon, when excited at 375 nm, had an emission maximum at 515 +/- 2 nm. The additi on of actin produced a nearly 70% increase in fluorescent intensity, a ccompanied by a blue shift in the emission maximum (i.e. lambda(em (ma x)) = 505 +/- 2 nm), suggesting that the probe now occupies a more non polar environment. Titration of labeled caldesmon with actin indicated a strong affinity (K-alpha = similar to 6 x 10(7) M(-1)). When actin was titrated with labeled caldesmon in the presence of caltropin in a 0.2 mM Ca2+ medium, its affinity for caldesmon was lowered (K-alpha = similar to 2 x 10(7) M(-1)). Caltropin, which is very effective in rev ersing caldesmon's inhibition of the actin-activated myosin ATPase (Ma ni, R. S., McCubbin, W. D., and Kay, C. M. (1992) Biochemistry 31, 118 96-11901), is shown in the present study to have a pronounced effect o n its binding to actin, suggesting a major role for caltropin in regul ating caldesmon in smooth muscle.