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

Calponin inhibits actomyosin Mg2+ ATPase and is proposed to regulate s mooth muscle contraction; however, the mechanism by which it exerts it s effect and the regulation of its behavior is still under investigati on. The proposed methods by which calponin regulation is effected incl ude reversible phosphorylation of calponin which would allow contracti on to occur and regulation by interaction with calcium-calmodulin. How ever, several investigators have been unable to find evidence of in vi vo phosphorylation of calponin, and the affinity between calponin and calmodulin is not high enough to suggest that this interaction is biol ogically 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 pr otein isolated from smooth muscle, is a dimer under native conditions and interacts with calponin in a calcium-dependent fashion in the rati o of 2 mol of dimer:1 mol of calponin. The formation of this complex c an be monitored by following the fluorescence of an acrylodan label on cysteine 273 of calponin, which undergoes a 35-nm blue shift in wavel ength peak from 505 to 470 nm when calponin becomes complexed with cal tropin. This fluorescence change when titrated with calcium indicates that the concentration of calcium required for complex formation is ap proximately 10(-5) M, corresponding to the low-affinity calcium-bindin g sites of caltropin. This complex was further characterized by circul ar dichroism (CD). The CD spectrum of the complex has a negative ellip ticity 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 i n alpha-helix and an increase in beta-sheet. Circular dichroism was us ed to monitor both the guanidine hydrochloride (Gdn.HCl) and temperatu re denaturation of the complex. In both cases, the results indicated t hat calponin is more stable in the complex than when free in solution. The midpoint for the guanidine titration curve of the complex was 2.4 8 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 do es not fully melt even at 80 degrees C. Caltropin is capable of regula ting calponin's inhibition of the actomyosin ATPase, and it does this more efficiently than calmodulin. Thus, calponin and caltropin have th e necessary properties to function as a regulatory complex in smooth m uscle contraction.