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

• Published in: The Journal of biological chemistry Vol. 265; no. 25; pp. 15231 - 15238
• Main Authors: BARTEGI, A, FATTOUM, A, DERANCOURT, J, KASSAB, R
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05-09-1990
• Subjects: actin; Actins - metabolism; Analytical, structural and metabolic biochemistry; Animals; Binding and carrier proteins; Binding Sites; Binding, Competitive; Biological and medical sciences; calmodulin; Calmodulin - metabolism; Calmodulin-Binding Proteins - isolation & purification; Calmodulin-Binding Proteins - metabolism; Chromatography, Affinity; Cyanogen Bromide; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Gizzard, Avian - metabolism; Kinetics; Molecular Weight; Muscle, Smooth - metabolism; Muscles - metabolism; Peptide Fragments - metabolism; Proteins; Rabbits; Tropomyosin - isolation & purification; Tropomyosin - metabolism; Turkeys; Binding protein; Vertebrata; Gizzard; Actins; Contractile protein; Dissociation constant; Molecular interaction; Binding site; Aves; Localization; Calmodulin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)77246-3

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.