SMOOTH-MUSCLE CALPONIN-CALTROPIN INTERACTION - EFFECT ON BIOLOGICAL-ACTIVITY AND STABILITY OF CALPONIN

Calponin inhibits actomyosin Mg2+ ATPase and is proposed to regulate smooth muscle contraction; however, the mechanism by which it exerts its effect and the regulation of its behavior is still under investigation. The proposed methods by which calponin regulation is effected include reversible phosphorylation of calponin which would allow contraction to occur and regulation by interaction with calcium-calmodulin. However, several investigators have been unable to find evidence of in vivo phosphorylation of calponin, and the affinity between calponin and calmodulin is not high enough to suggest that this interaction is biologically significant. In this paper, we present an alternative method of calponin regulation via calcium-caltropin and describe the calponin-caltropin complex for the first time. Caltropin, a calcium-binding protein isolated from smooth muscle, is a dimer under native conditions and interacts with calponin in a calcium-dependent fashion in the ratio of 2 mol of dimer:1 mol of calponin. The formation of this complex can be monitored by following the fluorescence of an acrylodan label on cysteine 273 of calponin, which undergoes a 35-nm blue shift in wavelength peak from 505 to 470 nm when calponin becomes complexed with caltropin. This fluorescence change when titrated with calcium indicates that the concentration of calcium required for complex formation is approximately 10(-5) M, corresponding to the low-affinity calcium-binding sites of caltropin. This complex was further characterized by circular dichroism (CD). The CD spectrum of the complex has a negative ellipticity 1590 degrees less than predicted for these two proteins in the presence of calcium, indicating secondary structure changes in one or both of the protein reactants, and these take the form of a decrease in alpha-helix and an increase in beta-sheet. Circular dichroism was used to monitor both the guanidine hydrochloride (Gdn.HCl) and temperature denaturation of the complex. In both cases, the results indicated that calponin is more stable in the complex than when free in solution. The midpoint for the guanidine titration curve of the complex was 2.48 M Gdn.HCl vs 1.25 M Gdn.HCl for calponin alone. The midpoint of the melting curve for calponin alone is 55 degrees C, while the complex does not fully melt even at 80 degrees C. Caltropin is capable of regulating calponin's inhibition of the actomyosin ATPase, and it does this more efficiently than calmodulin. Thus, calponin and caltropin have the necessary properties to function as a regulatory complex in smooth muscle contraction.