Characterization of the carboxyl-terminal 10-kDa cyanogen bromide fragment of caldesmon as an actin-calmodulin-binding region.

A pair of 10-kDa peptides, designated CB-a and CB-b, was isolated by calmodulin-Sepharose chromatography from a total CNBr digest of turkey gizzard caldesmon. CB-a encompasses the COOH-terminal segment of residues 659-756, according to the sequence of adult chicken gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R.G., and Lin, W.G. (1989) J. Biol. Chem. 264, 13873-13879), whereas CB-b comprises the same structure but was a few amino acids shorter at its COOH terminus. Both peptides cosedimented with F-actin, and their binding was increased by smooth muscle tropomyosin. The Kd values were 1.3 and 0.5 microM, in the absence and presence of tropomyosin, respectively, with a maximum binding capacity of 6.9 actins/mol of peptides. The CB-a/CB-b fragments inhibited, in a tropomyosin-sensitive and Ca2(+)-calmodulin-dependent manner, the skeletal actomyosin subfragment 1 ATPase activity to a level close but not identical to that observed for the parent caldesmon. Ca2(+)-calmodulin was selectively cross-linked to either caldesmon or the CNBr peptides with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide producing 1:1 covalent complexes that were retained neither by phenyl-Sepharose nor by immobilized calmodulin. Moreover, the cross-linked caldesmon bound weakly to F-actin and did not inhibit the actomyosin subfragment 1 ATPase in the absence of Ca2+. The results suggest that the CB-a/CB-b peptide region contains major regulatory determinants of caldesmon.